Optimized cellulase enzymes

ABSTRACT

The invention discloses cellulase enzymes with optimized properties for processing of cellulose- and lignocellulose-containing substrates. In particular, cellobiohydrolase enzymes with preferred characteristics are disclosed. The present invention provides fusion, insertion, deletion and/or substitution variants of such enzymes. Enzyme variants have enhanced thermostability, proteolytic stability, specific activity and/or stability at extreme pH. Nucleic acid molecules encoding said enzymes, a composition comprising said enzymes, a method for preparation, and the use for cellulose processing and/or for the production of biofuels are disclosed.

FIELD OF INVENTION

The invention discloses cellulase enzymes with optimized properties forprocessing of cellulose- and lignocellulose-containing substrates. Inparticular, cellobiohydrolase enzymes with preferred characteristics aredisclosed. The present invention provides fusion, insertion, deletionand/or substitution variants of such enzymes. Enzyme variants haveenhanced thermostability, proteolytic stability, specific activityand/or stability at extreme pH. Nucleic acid molecules encoding saidenzymes, a composition comprising said enzymes, a method forpreparation, and the use for cellulose processing and/or for theproduction of biofuels are disclosed.

BACKGROUND OF THE INVENTION

The development of production processes based on renewable resources ishighly desired, for example for the generation of ethanol fromcellulosic and lignocellulosic materials.

Cellulose material in pure form or in combination with hemicelluloseand/or lignin is a valuable and readily available raw material for theproduction of chemicals and fuels. A key step in processing celluloseand lignocellulose is the hydrolysis of the beta-1,4-linked glucosepolymer cellulose and the subsequent release of glucose monomers andshort glucose oligomers such as cellobiose, cellotriose, etc. Enzymesthat catalyze this reaction are found in various organisms, especiallyfilamentous fungi and bacteria, that are capable of degrading andhydrolysing cellulose.

Continuous processes for converting solid lignocellulosic biomass intocombustible fuel products are known. Treatment to make cellulosicsubstrates more susceptible to enzymatic degradation comprises milling,chemical processing and/or hydrothermal processing. Examples are wetoxidation and/or steam explosion. Such treatments increase theaccessibility of cellulose fibers and separate them from hemicelluloseand lignin.

A number of enzyme mixtures for hydrolysis of treated biomass are knownin the literature. Typically a mixture of endoglucanase, exoglucanaseand beta-glucosidase enzymes are required for the degradation ofcellulose polymers. Among these cellobiohydrolase (CBH) enzymes, andmore specifically cellobiohydrolase I (CBHI) enzymes, play a key role inthe hydrolysis step as they provide the most processive enzymaticactivity. CBHI enzymes catalyze the progressive hydrolytic release ofcellobiose from the reducing end of the cellulose polymers. (Lynd L R,Weimer P J, van Zyl W H, Pretorius I S. Microbial cellulose utilization:fundamentals and biotechnology. Microbiol Mol Biol Rev. 2002 September;66(3):506-77).

Hydrolyzed cellulosic materials contain several valuable carbohydratemolecules which can be isolated from the mixtures. Sugar containinghydrolysates of cellulosic materials can be used for microbialproduction of a variety of fine chemicals or biopolymers, such asorganic acids, ethanol or higher alcohols (also diols or polyols) orpolyhydroxyalkanoates (PHAs). One of the major uses of the sugarhydrolysates is in the production of biofuels.

Kurabi et al. (2005) describes preparations of cellulases fromTrichoderma reesei and other fungi, such as Penicillium sp. Theperformance has been analysed on steam-exploded and ethanolorganosolv-pretreated Douglas-fir. Better performance of enzyme mixturesappears to be a result of improved properties of single componentenzymes as well as the effect of each compound in the mixture,especially the presence of beta-glucosidase. (Kurabi A, Berlin A, GilkesN, Kilburn D, Bura R, Robinson J, Markov A, Skomarovsky A, Gusakov A,Okunev O, Sinitsyn A, Gregg D, Xie D, Saddler J. (2005) Enzymatichydrolysis of steam-exploded and ethanol organosolv-pretreatedDouglas-Fir by novel and commercial fungal cellulases. Appl BiochemBiotechnol. 121-124: 219-30).

Cellobiohydrolase sequences of the glucohydrolase class 7 (cel7) areknown to the art from several fungal sources. The Talaromyces emersoniiCel7 cellobiohydrolase is known and expression was reported inEscherichia coli (Grassick A, Murray P G, Thompson R, Collins C M,Byrnes L, Birrane G, Higgins T M, Tuohy M G. Three-dimensional structureof a thermostable native cellobiohydrolase, CBH IB, and molecularcharacterization of the cel7 gene from the filamentous fungus,Talaromyces emersonii. Eur J Biochem, 2004 November; 271(22):4495-506)and Saccharomyces cerevisiae (Voutilainen S P, Murray P G, Tuohy M G,Koivula A. Expression of Talaromyces emersonii cellobiohydrolase Cel7Ain Saccharomyces cerevisiae and rational mutagenesis to improve itsthermostability and activity. Protein Eng Des Sel. 2010 February;23(2):69-79), however the protein was either produced in inactive formor at rather low yields (less or equal to 5 mg/l). Hypocrea jecorinacellobiohydrolase I can be produced from wild type or engineered strainsof the genus Hypocrea or Trichoderma at high yields. Improved sequencesof Hypocrea jecorina Cel7A are disclosed by U.S. Pat. Nos. 7,459,299B2,7,452,707B2. WO2005/030926. WO01/04284A1 or US2009/0162916 A1.

Positions leading to improvements were deduced from alignments withsequences from reported thermostable enzymes, suggested from structuralinformation and shuffling of identified positions followed by limitedscreenings. Screening of larger libraries in transformable organismssuch as Saccharomyces cerevisiae was reported by application of verysensitive fluorescent substrates, which resemble native substrates in avery restricted way. (Percival Zhang Y H, Himmel M E, Mielenz J R.Outlook for cellulase improvement: screening and selection strategies.Biotechnol Adv. 2006 September-October; 24(5):452-81).

The production of cellobiohydrolases from other fungal systems such asThermoascus aurantiacus, Chrysosporium lucknowense or Phanerochaetechrysosporium was reported. Expression of Cel7 cellobiohydrolase fromyeasts was reported, but enzymatic yields or enzyme properties remainunsatisfactory. (Penttilä M E, André L, Lehtovaara P, Bailey M, Teeri TT, Knowles J K. Efficient secretion of two fungal cellobiohydrolases bySaccharomyces cerevisiae; Gene. 1988; 63(1):103-12).

WO03/000941 discloses a number of CBHs and their corresponding genesequences. Physiological properties and applications however were notdisclosed. The fusion of cellulose binding domains to catalytic subunitsof cellobiohydrolases is reported to improve the hydrolytic propertiesof proteins without a native domain.

US 2009042266 (A1) discloses fusions of Thermoascus aurantiacus Cel7Awith cellulose binding domains from cellobiohydrolase I from Chaetomiumthermophilum and Hypocrea jecorina.

U.S. Pat. No. 5,686,593 reports the fusion of specially designed linkerregions and binding domains to cellobiohydrolases.

Hong et al. (2003) describe the production of Thermoascus aurantiacusCBHI in yeast and its characterization. (Hong J, Tamaki H, Yamamoto K,Kumagai H Cloning of a gene encoding thermostable cellobiohydrolase fromThermoascus aurantiacus and its expression in yeast. Appl MicrobiolBiotechnol. 2003 November; 63(1):42-50).

Tuohy et al. (2002) report the expression and characterization ofTalaromyces emersonii CBH. (Tuohy M G, Walsh D J, Murray P G, ClaeyssensM, Cuffe M M, Savage A V, Coughlan M P.: Kinetic parameters and mode ofaction of the cellobiohydrolases produced by Talaromyces emersonii.Biochim Biophys Acta. 2002 Apr. 29; 1596(2):366-80).

Nevoigt et al. (2008) reports on the expression of cellulolytic enzymesin yeasts. (Nevoigt E. Progress in metabolic engineering ofSaccharomyces cerevisiae. Microbiol Mol Biol Rev. 2008 September;72(3):379-412).

Fujita et al. (2004) reports on a Saccharomyces cervisiae strainexpressing a combination of an endoglucanase, a beta glucosidase and aCBHII displayed on the cell surface. Cellobiohydrolase I (Cel7) was notused in this setup. (Fujita Y, Ito J, Ueda M, Fukuda H, Kondo A.Synergistic saccharification, and direct fermentation to ethanol, ofamorphous cellulose by use of an engineered yeast strain codisplayingthree types of cellulolytic enzyme. Appl Environ Microbiol. 2004February; 70(2):1207-12).

Boer H et al. (2000) describes the expression of GH7 classified enzymesin different yeast hosts but expressed protein levels were low. (Boer H,Teeri T T, Koivula A. Characterization of Trichoderma reeseicellobiohydrolase Cel7A secreted from Pichia pastoris using twodifferent promoters. Biotechnol Bioeng. 2000 Sep. 5; 69(5):486-94).

Godbole et al (1999) and Hong et al (2003) found that proteins of thisenzyme class expressed fom yeast were often misfolded, hyperglycosylatedand hydrolytic capabilities decreased compared to the protein expressedfrom the homologous host. (Godbole S, Decker S R, Nieves R A, Adney W S,Vinzant T B, Baker J O, Thomas S R, Himmel M E. Cloning and expressionof Trichoderma reesei cellobiohydrolase I in Pichia pastoris. BiotechnolProg. 1999 September-October; 15(5):828-33).

Kanokratana et al (2008), Li at al (2009) as well as CN01757710 describethe efficient expression of Cel7 CBH I enzymes, however these proteinsare lacking cellulose binding domains required for efficient substrateprocessing. (Kanokratana P, Chantasingh D, Champreda V, Tanapongpipat S,Pootanakit K, Eurwilaichitr L Identification and expression ofcellobiohydrolase (CBHI) gene from an endophytic fungus, Fusicoccum sp.(BCC4124) in Pichia pastoris. LProtein Expr Purif. 2008 March;58(1):148-53. Epub 2007 Sep. 19; Li Y L, Li H, Li A N, Li D C. Cloningof a gene encoding thermostable cellobiohydrolase from the thermophilicfungus Chaetomium thermophilum and its expression in Pichia pastoris. JAppl Microbiol. 2009 June; 106(6):1867-75).

Voutilainen (2008) and Viikari (2007) disclose Cel7 enzymes comprisingthermostable cellobiohydrolases, however with only low to moderateexpression levels from Trichoderma reesei. (Voutilainen S P, Puranen T,Siika-Aho M, Lappalainen A, Alapuranen M, Kallio J, Hooman S, Viikari L,Vehmaanperä J, Koivula A. Cloning, expression, and characterization ofnovel thermostable family 7 cellobiohydrolases. Biotechnol Bioeng. 2008Oct. 15; 101(3):515-28. PubMed PMID: 18512263; Viikari L, Alapuranen M,Puranen T, Vehmaanperä J, Siika-Aho M. Thermostable enzymes inlignocellulose hydrolysis. Adv Biochem Eng Biotechnol. 2007;108:121-45).

Grassick et al. (2004) disclose unfolded expression of CellobiohydrolaseI from Talaromyces emersonii in Escherichia coli but not in yeast.(Grassick A, Murray P G, Thompson R, Collins C M, Byrnes L, Birrane G,Higgins T M, Tuohy M G. Three-dimensional structure of a thermostablenative cellobiohydrolase, CBH IB, and molecular characterization of thecel7 gene from the filamentous fungus, Talaromyces emersonii. Eur JBiochem. 2004 November; 271(22):4495-506).

Therefore, there is a need for cellulase enzymes with improvedcharacteristics for the use in technical processes for cellulosehydrolysis. In particular there is a need for CBH enzymes with highercatalytic activity and/or higher stability under process conditions.Moreover there is a need for CBH enzymes with higher productivity infungal and/or yeast expression and secretion systems.

SUMMARY OF THE INVENTION

The present invention provides a polypeptide having cellobiohydrolaseactivity. In a preferred embodiment, the invention provides athermostable polypeptide having cellobiohydrolase activity. That is, inthis embodiment, the polypeptide maintains 50% of its maximum substrateconversion capacity when the conversion is done for 60 minutes at 60° C.or higher, preferably 62° C. or higher, and in a particular embodiment64° C. or higher, such as 66° C. or higher. This polypeptide comprisesan amino acid sequence with at least 54%, preferably at least 56%, morepreferably at least 58%, particularly preferably at least 60%, such asat least 62%, particularly at least 64%, such as at least 66%, and mostpreferably preferably at least 68% sequence identity to SEQ ID NO5. Theinvention also provides a polypeptide which comprises an amino acidsequence with at least 85% sequence identity to SEQ ID NO: 2.

Furthermore, the present invention discloses a nucleic acid encoding thepolypeptide of the present invention, preferably having at least 95%identity to SEQ ID NO: 1. a vector comprising this nucleic acid and ahost transformed with said vector.

The present invention further provides a method of producing acellobiohydrolase protein encoded by a vector of the present invention,a method for identifying polypeptides having cellobiohydrolase activity,and a method of preparing such polypeptides having cellobiohydrolaseactivity. It also provides a method of identifying such polypeptideswhich maintain 50% or more of maimum substrate conversion capacity atelevated temperatures, such as at 60° C. or more.

The present invention also provides a polypeptide havingcellobiohydrolase activity, wherein the polypeptide comprises an aminoacid sequence having at least 85% sequence identity to SEQ ID NO: 2wherein one or more specific amino acid residues of the sequence definedby SEQ ID NO: 2 are modified by substitution or deletion, as well asinsertion mutants. Examples of such mutants include Q1. G4. A6. T15.Q28. W40. D64. E65. A72. S86. K92, V130. V152. Y155. K159. D181. E183.N194. D202. P224. T243. Y244. I277. K304. N310, S311. N318. D320. T335.T344. D346. Q349. A358. Y374. A375. T392. T393. D410. Y422, P442. N445.R446. T456. S460. P462. G463. H468 and/or V482 of amino acids 1 to 500of SEQ ID NO: 2. but the invention is by no means limited to these.Further specific positions are given below.

Moreover, the present invention provides a polypeptide havingcellobiohydrolase activity, which is obtainable by the method ofpreparing a polypeptide having cellobiohydrolase activity according tothe present invention, and a polypeptide having cellobiohydrolaseactivity, wherein the polypeptide comprises an amino acid sequencehaving at least 80% sequence identity to SEQ ID NO: 5. wherein one ormore of the following amino acid residues of the sequence defined by SEQID NO: 5 are modified by substitution or deletion, as well as insertionmutants. Examples of such mutants include Q1. G4. A6. T15. Q28. W40,D64. E65. A72. S86. K92. V130. V152. Y155. K159. D181. E183. N194. D202.P224. T243, Y244. I277. K304. N310. S311. N318. D320. T335. T344. D346.Q349. A358. Y374. A375, T392. T393. D410 and/or Y422 of amino acids 1 to440 of SEQ ID NO: 5. but the invention is by no means limited to these.Further specific positions are given below.

The present invention furthermore provides a polypeptide havingcellobiohydrolase activity comprising an amino acid sequence having atleast 85% sequence identity to SEQ ID NO: 12 wherein one or more of thefollowing amino acid residues of the sequence defined by SEQ ID NO: 12are modified by substitution or deletion as well as insertion mutants.Examples of such mutants include Q1. T15. Q28. W40. C72. V133. V155.Y158. T162. Y247, N307. G308. E317. S341. D345. Y370. T389. Q406. N441.R442. T452. S456. P458. G459, H464 and/or V478. but the invention is byno means limited to these. Further specific positions are given below.

The present invention further provides the use of a polypeptide or thecomposition of the present invention for the enzymatic degradation oflignocellulosic biomass, and/or for textiles processing and/or asingredient in detergents and/or as ingredient in food or feedcompositions.

BRIEF DESCRIPTION OF THE FIGURES

FIG. 1: Restriction Maps of pV1 for constitutive expression of Proteinsin Pichia pastoris: pUC19—ori: Origin of replication in E. coli; KanR:Kanamycine/G418 Resistance with TEF1 and EMZ Promoter sequences forselection in Pichia pastoris and E. coli. respectively; 5′GAP:glyceraldehyde-3-phosphate dehydrogenase Promoter region; 3′-GAP:terminator region; SP MFalpha: Saccharomyces cerevisiae mating factoralpha signal sequence; MCS: multiple cloning site.

FIG. 2: Commassie stained SDS-PAGE of 10-fold concentrated supernatantsof shake-flask cultures of Pichia pastoris CBS 7435 containingexpression plasmids with coding sequences for the mature CBHI proteinsof Trichoderma viride (CBH-f; lane 1), Humicola grisea (CBH-d; lane 2),Talaromyces emersonii (CBH-b; lane 3), Thermoascus aurantiacus (CBH-e;lane 4), as well as the Talaromyces emersonii CBHI-CBD fusion (CBH-a;lane 6) and the Humicola grisea-CBD fusion (CBH-g; lane 7) in N-terminalfusion to the signal peptide of the Saccharomyces cerevisiae matingfactor alpha under control of the Pichia pastorisglyceraldehyde-3-phosphate dehydrogenase promoter

FIG. 3: Map of the pV3 expression plasmid for protein expression inPichia pastoris. Replicons: pUC19—ori: Origin of replication in E. coli;ZeoR: Zeocine resistance gene with TEF1 and EM7 Promoter promotersequences for expression in Pichia pastoris and E. coli, respectively;AOX I promoter: Promoter region of the Pichia pastoris alcohol oxidase Igene; AOX 1 transcriptional terminator: terminator region; SP MFalpha:Saccharomyces cerevisiae mating factor alpha signal sequence; MCS:multiple cloning site.

FIG. 4: SDS-PAGE analysis of culture supernatant samples taken from thefermentation of a Pichia pastoris strain with a genomic integration ofan AOXI-expression cassette, expressing the Talaromyces emersoniiCBHI/Trichoderma reesei-CBD fusion peptide (CBH-a) in a 7I bioreactorduring methanol induction. Samples P1-P7 were are taken at the beginningof the methanol induction and after 20, 45, 119.5, 142.5, 145.5 and 167hours, respectively.

FIG. 5: Map of pV4 expression plasmid for the expression of theTalaromyces emersonii CBHI/Trichoderma reesei-CBD fusion peptide(CBH-ah) in Trichoderma reesei. Replicon: pUC19 for replication in E.coli. cbh1 5′: 5′ promoter region of the Trichoderma CBHI gene; cbh1signal peptide: Coding sequence for the Trichoderma reesei CBHI leaderpeptide; CBH-a: Talaromyces emersonii CBHI/Trichoderma reesei-CBD fusionpeptide: coding region for SeqID NO. 18; cbh1 Terminator: 3′ terminationregion of the Trichoderma reesei CBHI locus; hygromycine resistance:coding region of the hygromycine phosphotransferase under control of aTrichoderma reesei phosphoglycerate kinase promoter; cbh1 3′: homologysequence to the termination region of the Trichoderma reesei CBHI locusfor double crossover events.

FIG. 6: SDS-Page of Trichoderma reesei culture supernatants. Lane 1shows the expression pattern of a replacement strain carrying aTalaromyces emersonii CBHI/Trichoderma reesei-CBD fusion (CBH-ah) inplace of the native CBHI gene. In comparison lane 2 shows the patternfor the unmodified strain under same conditions. M: molecular sizemarker

FIG. 7: Determination of IT50 values from Substrate Conversion Capacityvs. temperature graphs after normalization. For the normalization stepthe maximum and the minimum fluorescence values for the selectedtemperature are correlated to 1 or 0. respectively. Linear interpolationto F′(T)=0.5 between the nearest two temperature points with normalizedvalues next to 0.5 results in the defined IT50 temperature.

FIG. 8: Normalized Conversion Capacity vs. temperature graphs of “wt”Talaromyces emersonii CBHI/Trichoderma reesei-CBD fusions (CBH-ah: SeqIDNO. 18=SeqID NO. 2+6× His-Tag) and mutants based on4-Methylumbelliferyl-β-D-lactoside hydrolysis results evaluated atvarious temperatures. The fluorescence values were normalized accordingto FIG. 8 over the temperature range from 55° C. to 75° C.

A . . . wt;

B . . . G4C,A72C;

C . . . G4C,A72C,Q349K;

D . . . G4C,A72C,D181N,Q349K;

E . . . Q1L,G4C,A72C,D181N,E183K,Q349R;

F . . . Q1L,G4C,A72C,S86T, D181N,E183K,D320V,Q349R;

G . . . G4C, A72C,E183K,D202Y,N310D,Q349R;

H . . . Q1L,G4C,A72C, A145T,H203R,Q349K,T403K;

I . . . , Q1L, G4C, Q28R, E65V, A72C, D181N, E183M, S192S, P224L, S311G,D320I, D346E, Q349K, T393V, Y422F, P442S, N445D, R446G, H468L, V482I

J . . . , Q1L, G4C, Q28K, E65K, A72C, L119L, D181N, E183M, S192S, S311G,N318Y, D320I, T335I, D346E, Q349K, T393V, Y422F, N445D, R446G, H468Q,V482T

K . . . , Q1L, G4C, Q28K, E65V, A72C, L119L, D181N, E183M, S192S, D202N,P224L, S311G, T335I, D346E, Q349K, T393V, Y422F, P442S, N445D, R446G,H468Q, V482I

L . . . Q1L, G4C, Q28R, E65V, A72C, G151GCGRSG, D181N, E183M, P224L,S311G, D320I, D346E, Q349K, T393V, Y422F, P442S, N445D, R446G, H468L,V482I

M . . . Q1L, G4C, Q28R, E65V, A72C, K159KCGRNK, D181N, E183M, P224L,S311G, D320I, D346E, Q349K, T393V, Y422F, P442S, N445D, R446G, H468L,V482I

FIG. 9: Glucose yields of hydrolysis of pretreated straw with wt andmutated Talaromyces emersonii CBHI/Trichoderma reesei-CBD (CBH-ah)fusion protein after hydrolysis for 48 hours in the presence of aβ-glycosidase. The variants are characterized by the following mutationswith respect to SeqID NO. 18 and were expressed from Pichia pastoris inshake flask cultures and isolated from the supernatant by affinitychomatography using Ni-NTA.

A: wt

B: G4C, A72C

C: G4C, A72C, Q349R

D: Q1L, G4C, A72C, D181N, E183K, Q349R

E: Q1L, G4C, Q28R, E65V, A72C, D181N, E183M, P224L, S311G, D320I, D346E,Q349K, T393V, Y422F, P442S, N445D, R446G, H468L, V482I

F: Q1L, G4C, Q28R, E65V, A72C, G151GCGRSG, D181N, E183M, P224L, S311G,D320I, D346E, Q349K, T393V, Y422F, P442S, N445D, R446G, H468L, V482I

FIG. 10: Alignment of SeqID. NO 2 with the Trichoderma reesei CBHI. Thealignment matrix blosum62mt2 with gap opening penalty of 10 and gapextension penalty of 0.1 was used to create the alignment.

DETAILED DESCRIPTION OF THE INVENTION

The present invention provides polypeptides having cellobiohydrolaseactivity. In a preferred embodiment, the invention provides athermostable polypeptide having cellobiohydrolase activity. In apreferred aspect, the invention discloses protein variants that show ahigh activity at high temperature over an extended period of time.Preferably, the polypeptide of the present invention maintains 50% ofits maximum substrate conversion capacity when the conversion is donefor 60 minutes at a temperature of 60° C. or higher. The respectivetemperature is also referred to as the IT50 value. In other words, theIT50 value is preferably 60° C. or higher, but more preferably 62° C. orhigher. That is, in this embodiment, the polypeptide maintains 50% ofits maximum substrate conversion capacity when the conversion is donefor 60 minutes at 60° C. or higher, preferably 62° C. or higher, and ina particular embodiment 64° C. or higher, such as 66° C. or higher.Furthermore, the polypeptides of the present invention have preferablyan IT50 value in the range of 62 to 80° C., more preferably 65 to 75° C.

“Substrate Conversion Capacity” of an enzyme is herein defined as thedegree of substrate conversion catalyzed by an amount of enzyme within acertain time period under defined conditions (Substrate concentration,pH value and buffer concentration, temperature), as can be determined byend-point assaying of the enzymatic reaction under said conditions.“Maximum Substrate Conversion Capacity” of an enzyme is herein definedas the maximum in Substrate Conversion Capacity found for the enzymewithin a number of measurements performed as described before, whereonly one parameter, e.g. the temperature, was varied within a definedrange. According to the present invention, the assay described inExample 8 is used to determine these parameters.

This polypeptide comprises an amino acid sequence with at least 54%,preferably at least 56%, more preferably at least 58%, particularlypreferably at least 60%, such as at least 62%, particularly at least64%, such as at least 66%, and most preferably preferably at least 68%sequence identity to SEQ ID NO: 5. The term “Identity over a sequencelength of y residues” (wherein y is any Integer, such as, asillustrative example, 200, 255, 256, 300, 400, 437, 500) means that y isa—preferably continuous—portion of the parenteral sequence (in thisparticular case SEQ ID NO: 5. but the same is true throughout thisapplication, also with respect to other, specifically indicatedparenteral sequences which which the sequences of this invention may becompared) which is used as a basis for the comparison of sequenceidentity. Thus, for the comparison of sequence identity (sequencealignment), preferably 200 or more, more preferably 300 or more, evenmore preferably 400 or more, and most preferably 437 positions of theparental sequence given in SEQ ID NO: 5 are taken into consideration.The details of how the percentages of sequence identities are calculatedare given below. It should also be noted, that, unless explicitlyotherwise specified in this specification, the entire sequence of theparental sequence (such as, in this particular case, SEQ ID NO:5) (i.e.from the first to the last amino acid residue) shall be used as a parentsequence.

In a preferred embodiment, the polypeptide capable of maintaining 50% ofits maximum substrate conversion capacity when the conversion is donefor 60 minutes at 60° C. or higher, preferably 62° C. or higher, is apolypeptide which differs from SEQ ID NO: 5 by at least one mutation,whereby the mutation may be an insertion, deletion or substitution ofone or more amino acid residues. Also preferred are at least two suchmutations, such as at least 4. at least 5. at least 6. at least 7. atleast 10 such mutations with respect to the polypeptide given in SEQ IDNO: 5.

“Cellobiohydrolase” or “CBH” refers to enzymes that cleave cellulosefrom the end of the glucose chain and produce cellobiose as the mainproduct. Alternative names are 1,4-beta-D-glucan cellobiohydrolases orcellulose 1,4-beta-cellobiosidases. CBHs hydrolyze the1,4-beta-D-glucosidic linkages from the reducing or non-reducing ends ofa polymer containing said linkages. “Cellobiohydrolase I” or “CBH I” actfrom the reducing end of the cellulose fiber. “Cellobiohydrolase II” or“CBH II” act from the non-reducing end of the cellulose fiber.Cellobiohydrolases typically have a structure consisting of a catalyticdomain and one or more “cellulose-binding domains” or “CBD”. Suchdomains can be located either at the N- or C-terminus of the catalyticdomain. CBDs have carbohydrate-binding activity and they mediate thebinding of the cellulase to crystalline cellulose and presence orabsence of binding domains are known to have a major impact on theprocessivity of an enzyme especially on polymeric substrates.

In a preferred embodiment, this polypeptide is further characterized bycomprising an amino acid sequence having at least 80% sequence identityto SEQ ID NO:5. more preferably at least 85% sequence identity to SEQ IDNO:5. such as at least 90% sequence identity to SEQ ID NO:5. and mostpreferably at least 95% sequence identity to SEQ ID NO.5.

The polypeptide having cellobiohydrolase activity as defined above is,in an even more preferred embodiment, further characterized as follows:It is the polypeptide as defined above, wherein one or more of the aminoacid residues of the sequence defined by SEQ ID NO: 5 are modified bysubstitution or deletion at one or more positions which are preferablyselected from

Q1. Q2. G4. A6. T7. A8. N10. P12. T15. A21. G23. S24. T26. T27. Q28.N29. G30. A31, V32. N37. W40. V41. G46. Y47. T48. N49. C50. T52. N54.D57. T59. Y60. D64. E65. A68, Q69. A72. V84. S86. S89. S90. K92. S99.Q109. D110. D111. I116. F117. K118. L119. L120, D129. V130. G139. A145.M146. V152. K154. Y155. N157. N158. K159. K163. G167. Q172, F179. I180.D181. E183. E187. G188. Q190. S192. S193. N194. I200. D202. H203. D211,V212. A221. P224. D228. T229. G231. T233. M234. S236. T243. Y244. S245.N246. D247, G251. F260. G266. K275. I276. I277. T280. L290. D293. G294.T295. T297. T299. S301, K304. F306. N310. S311. V313. I314. N318. D320.I321. T325. N327. T335. A340. F341, D343. T344. D345. D346. Q349. H350.A354. K355. A358. Q361. Q362. G363. M364. V367, D373. Y374. A375. A376.P386. T387. D390. T392. T393. P394. T400. P402. T403. D404, D410. N417.S418. T421. Y422 and/or one or more insertions after positions G151.K159,

and in a more preferred embodiment are modified by substitution ordeletion at one or more positions selected from Q1. Q2. G4. A6. T7. A8.N10. A21. S24. T26, 127. Q28. N29. G30, W40. Y47, 064. E65. A68. Q69.A72. S86. K92. K118. Y155. D181. E183. Q190. S192, N194. D202. H203.P224. T229. G231. M234. S236. T243. D247. S311. N318. D320. T335, A340.T344. D346. Q349. K355. Y374. A375. T387. D390. T392. T393. Y422 and/orone or more insertion of 1-8 amino acids after positions G151. K159

and in an even more preferred embodiment are modified by substitution ordeletion at one or more positions selected from Q1. Q2. G4. A6. T7. A8.N10. Q28. E65. A72. S86. D181, E183. D202. P224. S311. N318. D320. T335.D346. Q349. T392. T393. Y422 and/or insertions at one or more afterpositions and/or one or more insertion of 5 amino acids after positionsG151. K159 of of amino acids 1 to 437 of SEQ ID NO: 5.

Also preferred are embodiments wherein the respective mutation(s) givenabove is (are) introduced into the polypeptide given in SEQ ID NO: 2. asoutlined below.

The skilled person will understand that several of these given mutationscan be combined with each other, i.e. that a polypeptide havingcellobiohydrolase activity, where, for example Q69 and T232 aresubstituted for other amino acid residues, is comprised in thisembodiment. The term “insertion after position(s) x” is to be understoodthat the insertion may be at any position which is on the C-terminalside (closer to the C-terminus) of the position x; however, insertionsimmediately following the position x are strongly preferred (wherein xis any position).

The present invention also discloses a polypeptide havingcellobiohydrolase activity, which comprises an amino acid sequence withat least 85% sequence identity to SEQ ID NO: 2. It is preferred thatthis polypeptide with at least 85% sequence identity to SEQ ID NO: 2 isa polypeptide which has also a degree of identity with SEQ ID NO: 5 asgiven above, such as having at least 60% (or more, see above) sequenceidentity with the polypeptide given in SEQ ID NO: 5. and/or any one ormore of the more particular identity embodiments of percentage identityto SEQ ID NO: 5 as given in detail above. Thus, the polypeptide havingat least 85% sequence identity to SEQ ID NO: 2 is an embodiment which iscomprised in the invention relating to a polypeptide having at least 60%sequence identity with the polypeptide given in SEQ ID NO: 5. Theskilled person will readily recognize the common inventive conceptunderlying the thermostable variants of SEQ ID NO: 2 and SEQ ID NO: 5.

Equivalent to what has been said above for SEQ ID NO: 5. for thesequence alignment of SEQ ID NO: 2 as defined herein, preferably 200 ormore, more preferably 300 or more, even more preferably 400 or more, andmost preferably 437 positions of SEQ ID NO: 5 are taken intoconsideration. The details of how the percentages of sequence identitiesare calculated are given below. In a more preferred embodiment, therespective polypeptide comprises an amino acid sequence having at least85% sequence identity to SEQ ID NO: 2 over a sequence length of 500amino acid residues. Even more preferably, the present inventioncomprises an amino acid sequence having at least 90%, or even morepreferably of at least least 95% or 98% sequence identity to SEQ ID NO:2 over a sequence length of 500 amino acid residues.

The parental sequence is given in SEQ ID NO: 2. The sequence derivesfrom the C-terminal fusion of the linker domain and cellulose bindingdomain of Trichoderma reesei CBHI (SEQ ID NO: 4) to the catalytic domainof Talaromyces emersonii CBHI (SEQ ID NO: 5).The invention furthercomprises other fusion proteins comprising any cellulose binding domainand a derivative of the catalytic domain of Talaromyces emersonii CBHI(SEQ ID NO: 5), preferably with the temperature stabilitycharacteristics given above. The cellulose binding domain may be fromany source. The polypeptides according to the invention may additionallycarry a hexahistidine tag. Thus, by means of illustration, variants ofany one of the polypeptides shown in SEQ ID NO: 42, 44, 46, 48 or 50 areincluded in this invention. The variants are preferably such that thepolypeptides exhibit temperature stability, as described and definedabove.

The polypeptide of the present invention preferably comprises an aminoacid sequence having at least 90%, preferably at least 95%, morepreferably at least 99% sequence identity to SEQ ID NO: 2. Furthermore,it is particularly preferred that the amino acid sequence of thepolypeptide has the sequence as defined by SEQ ID NO: 2. or a sequenceas defined by SEQ ID NO: 2 wherein 1 to 75. more preferably 1 to 35amino acid residues are substituted, deleted, or inserted.

Particularly preferred are variants of the protein of SEQ ID NO: 2. SEQID NO: 5 or SEQ ID NO: 12. “Protein variants” are polypeptides whoseamino acid sequence differs in one or more positions from this parentalprotein, whereby differences might be replacements of one amino acid byanother, deletions of single or several amino acids, or insertion ofadditional amino acids or stretches of amino acids into the parentalsequence. Per definition variants of the parental polypeptide shall bedistinguished from other polypeptides by comparison of sequence identity(alignments) using the ClustalW Algorithm (Larkin M. A., BlackshieldsG., Brown N. P., Chenna R., McGettigan P. A., McWilliam H., Valentin F.,Wallace I. M., Wilm A., Lopez R., Thompson J. D., Gibson T. J. andHiggins D. G. (2007) ClustalW and ClustalX version 2. Bloinformatics2007 23(21): 2947-2948). Methods for the generation of such proteinvariants include random or site directed mutagenesis, site-saturationmutagenesis, PCR-based fragment assembly, DNA shuffling, homologousrecombination in-vitro or in-vivo, and methods of gene-synthesis.

The nomenclature of amino acids, peptides, nucleotides and nucleic acidsis done according to the suggestions of IUPAC. Generally amino acids arenamed within this document according to the one letter code.

Exchanges of single amino acids are described by naming the singleletter code of the original amino acid followed by its position numberand the single letter code of the replacing amino acid, i.e. the changeof glutamine at position one to a leucine at this position is describedas “Q1L”. For deletions of single positions from the sequence the symbolof the replacing amino acid is substituted by the three letterabbreviation “del” thus the deletion of alanine at position 3 would bereferred to as “A3del”. Inserted additional amino acids receive thenumber of the preceding position extended by a small letter inalphabetical order relative to their distance to their point ofinsertion. Thus, the insertion of two tryptophanes after position 3 isreferred to as “3aW, 3bW” or simply as A3AWW (i.e formal replacement of“A” at position 3 by the amino acid residues “AWW”). Introduction ofuntranslated codons TAA, TGA and TAG into the nucleic acid sequence isindicated as “*” in the amino acid sequence, thus the introduction of aterminating codon at position 4 of the amino acid sequence is referredto as “G4*”.

Multiple mutations are separated by a plus sign or a slash or a comma.For example, two mutations in positions 20 and 21 substituting alanineand glutamic acid for glycine and serine, respectively, are indicated as“A20G+E21S” or “A20G/E21S” “A20G,E21S”.

When an amino acid residue at a given position is substituted with twoor more alternative amino acid residues these residues are separated bya comma or a slash. For example, substitution of alanine at position 30with either glycine or glutamic acid is indicated as “A20G,E” or“A20G/E”, or “A20G, A20E”.

When a position suitable for modification is identified herein withoutany specific modification being suggested, it is to be understood thatany amino acid residue may be substituted for the amino acid residuepresent in the position. Thus:for instance, when a modification of analanine in position 20 is mentioned but not specified; it is to beunderstood that the alanine may be deleted or substituted for any otheramino acid residue (i.e. any one of R, N, D, C, Q, E, G, H, I, L, K, M,F, P, S, T, W, Y and V).

The terms “similar mutation” or “similar substitution” refer to an aminoacid mutation that a person skilled in the art would consider similar toa first mutation. Similar in this context means an amino acid that hassimilar chemical characteristics. If, for example, a mutation at aspecific position leads to a substitution of a non-aliphatic amino acidresidue (e.g. Ser) with an aliphatic amino acid residue (e.g. Leu),then' a substitution at the same position with a different aliphaticamino acid (e.g. Ile or Val) is referred to as a similar mutation.Further amino acid characteristics include size of the residue,hydrophobicity, polarity, charge, pK-value, and other amino acidcharacteristics known in the art. Accordingly, a similar mutation mayinclude substitution such as basic for basic, acidic for acidic, polarfor polar etc. The sets of amino acids thus derived are likely to beconserved for structural reasons. These sets can be described in theform of a Venn diagram (Livingstone C D. and Barton G J. (1993) “Proteinsequence alignments: a strategy for the hierarchical analysis of residueconservation” Comput. Appl Biosci. 9: 745-756; Taylor W. R. (1986) “Theclassification of amino acid conservation” J. Theor. Biol. 119;205-218). Similar substitutions may be made, for example, according tothe following grouping of amino acids: Hydrophobic: F W Y H K M I L V AG; Aromatic: F W Y H; Aliphatic: I L V; Polar: W Y H K R E D C S T N;Charged H K R E D; Positively charged: H K R; Negatively charged: E D.

As convention for numbering of amino acids and designation of proteinvariants for the description of protein variants the first glutamine (Q)of the amino acid sequence QQAGTA within the parental protein sequencegiven in SEQ ID NO: 2 is referred to as position number 1 or Q1 orglutamine 1. The numbering of all amino acids will be according to theirposition in the parental sequence given in SEQ ID NO: 2 relative to thisposition number 1.

The present invention furthermore discloses specific variants of thepolypeptides of the present invention as given above, such as variantsof SEQ ID NO: 2. with changes of their sequence at one or more of thepositions given hereafter. I.e., the invention provides, in a particularembodiment, the polypeptide as above, wherein one or more of thefollowing amino acid residues of the sequence defined by SEQ ID NO: 2are preferably modified by substitution or deletion at positions Q1. Q2.G4. A6. T7. A8. N10. P12. T15. A21. G23. S24, T26. T27. Q28. N29. G30.A31. V32. N37; W40. V41. G46. Y47. T48. N49. C50. T52. N54, D57. T59.Y60. D64. E65. A68. Q69. A72. V84. S86. S89. S90. K92. S99. Q109. D110,D111. I116. F117. K118. L119. L120. D129. V130. G139. A145. M146. V152.K154. Y155, N157. N158. K159. K163. G167. Q172. F179. I180. D181. E183.E187. G188. Q190. S192, S193. N194. I200. D202. H203. D211. V212. A221.P224. D228. T229. G231. T233. M234, S236. T243. Y244. S245. N246. D247.G251. F260. G266. K275. I276. I277. T280. L290, D293. G294. T295. T297.T299. S301. K304. F306. N310. S311. V313. I314. N318. D320, I321. T325.N327. T335. A340. F341. D343. T344. D345. D346. Q349. H350. A354. K355,A358. Q361. Q362. G363. M364. V367. D373. Y374. A375. A376. P386. T387.D390. T392, T393. P394. T400. P402. T403. D404. D410. N417. S418. T421.Y422. F427. P429. I430, G431. T433. G434. N435. P436. S437. G439. N440.P441. P442. G443. N445. R446. T448, T449. T450. T451. R453. P454. A455.T456. T457. G459. S460. S461. P462. G463. P464, T465. S467. H468. G470.C472. G474. G476. Y477. S478. P480. V482. C483. S485. G486, T488. C489.Q490. V491. L492. N493. Y495. Y496. Q498. C499. L500 and/or by one ormore insertions after positions G151. K159. G434. A455 or P464; of aminoacids 1 to 500 of SEQ ID NO: 2.

In a more preferred embodiment one or more of the following amino acidresidues of the sequence defined by SEQ ID NO: 2 are preferably modifiedby substitution or deletion at positions selected from Q1. Q2. G4. A6.T7. A8. N10. A21. S24. T26. T27. Q28. N29. G30, W40. Y47. D64. E65. A68.Q69. A72. S86. K92. K118. Y155. D181. E183. Q190. S192, N194. D202.H203. P224. T229. G231. M234. S236. T243. D247. S311. N318. D320. T335,A340. T344. D346. Q349. K355. Y374. A375. T387. D390. T392. T393. Y422.P436. P442, N445. R446. T448. T451. R453. P462. G463. H468. P480. V482.S485. and/or by one or more insertion of 1-8 amino acids after positionsG151. K159. G434. A455 or P464. and in an even more preferred embodimentare modified by substitution or deletion at one or more positionsselected from Q1. Q2. G4. A6. T7. A8. N10. Q28. E65. A72. S86. D181.E183, D202. P224. S311. N318. D320. T335. D346. Q349. T392. T393. Y422.P442. N445. R446, H468. V482. and/or by insertions at one or more afterpositions and/or one or more insertion of 5 amino acids after positionsG151. K159. G434. A455 or P464 of amino acids 1 to 500 of SEQ ID NO: 2.

Also comprised in the invention are the respective mutations at any oneor more of the specified mutations 1 to 430 of SEQ ID NO: 5. The skilledperson will recognize that residues 1 to 430 of SEQ ID NO: 5 areequivalent to positions 1 to 430 of SEQ ID NO2. and can thereforereadily transfer the detailed teaching given above and below forpreferred modifications of SEQ ID NO: 2 for any one or more of positions1 to 430 to the respective one or more position (1 to 430) of SEQ IDNO5. As an illustrative and non-limiting example, it is apparent for theskilled person that, since D390 is one particular position at which amodification in SEQ ID NO: 2 is preferred, D390 is likewise a positionat which a modification in SEQ ID NO: 5 is preferred.

In a preferred embodiment, the variant of the polypeptide of the presentinvention is a polypeptide as described above, wherein specifically oneor more of the following amino acid residues of the sequence defined bySEQ ID NO: 2 are modified as shown in Table 1. Shown are preferred, morepreferred and most preferred modification. Any of these mutations may becombined, with each other. However, in a particular embodiment it ispreferred that the mutations are selected only among the more preferredand most preferred embodiments shown in Table 1. Even more preferably,only modifications indicated as most preferred are chosen. The skilledperson will be aware that any several such mutations are combinable witheach other.

TABLE 1 Preferred exchanges and similar mutations Most PositionPreferred More Preferred Preferred Q1 L L L Q2 P, S P, S S G4 C C C A6G, L, V G, L, V L T7 Q Q Q A8 S S S N10 T, D T, D T, D P12 Q T15 S A21S, T, C S, T, C G23 A, D, N S24 T, C, N T, C, N T26 I, N I, N T27 S, QS, Q Q28 L, K, R, N L, K, R, N K, R N29 T, Y T, Y G30 A A A31 S V32 GN37 S W40 R R V41 T G46 S Y47 S, F S, F T48 A N49 S C50 S T52 D N54 SD57 S T59 M Y60 H D64 N N E65 V, M, K V, M, K V, M, K A68 T T Q69 K, RK, R A72 V, C V, C C V84 A S86 T T T S89 N S90 T, F K92 R R S99 T Q109 RD110 G, S, N D111 H, E I116 V, K, E F117 Y K118 A, T, Q A, T, Q L119 L,I L120 P, M D129 N V130 I G139 S A145 T M146 C G151 GCGRSG GCGRSG GCGRSGV152 A, E K154 R Y155 S, C, H S, C, H N157 S N158 D K159 E, KCGRNKKCGRNK KCGRNK K163 C G167 C Q172 Q F179 I I180 N D181 N N N E183 V, M, KV, M, K V, M, K E187 K G188 C Q190 L, K L, K S192 L, I, P, T, M L, I, P,T, M S193 L, P, T N194 G, L, I, V, S, C, K, G, L, I, V, S, C, R, D, Q, YK, R, D, Q, Y I200 N, F D202 G, I, V, N, F, Y G, I, V, N, F, Y G, I, V,N, F, Y H203 R R D211 G V212 L A221 V P224 L L L D228 N T229 A, S, M A,S, M G231 D D T233 S M234 L, I, V, T, K L, I, V, T, K S236 F, Y F, YT243 G, A, L, I, V, P, S, C, G, A, L, I, V, P, S, C, M, R, D, Q, F, Y, WM, R, D, Q, F, Y, W Y244 H, F S245 T N246 S, K, D D247 N N G251 R F260 CG266 S K275 E I276 V I277 V T280 A L290 H D293 R, H G294 A T295 S T297 NT299 I, S S301 C K304 R F306 L, Y N310 D, E S311 G, D, N G, D, N G, D, NV313 I I314 F N318 I, H, D, Y I, H, D, Y I, D, Y D320 I, V, E, N I, V,E, N I, V, N I321 N T325 A, I N327 Y T335 I I I A340 G, S, T G, S, TF341 C D343 A T344 M M D345 E D346 G, A, V, E G, A, V, E G, A, V, E Q349K, R K, R K, R H350 Y A354 T K355 Q Q A358 E Q361 R Q362 G, R, H G363 PM364 L, S V367 A D373 E Y374 A, P, S, C, R, H, D A, P, S, C, R, H, DA375 G, L, V, T, C, M, R, G, L, V, T, C, M, R, D, E, N, Q, Y D, E, N, Q,Y A376 T P386 L, S T387 A, S A, S D390 G, E G, E T392 S, M, K S, M, K MT393 A, I, V, S A, I, V, S A, I, V P394 C T400 S P402 S T403 K D404 ND410 G N417 Y S418 P T421 I Y422 F F F F427 Y P429 C I430 L G431 D T433S, E G434 S, GAAATG GAAATG GAAATG N435 Q P436 S S S437 P G439 V, D N440E P441 A, L, S P442 S, Q, del S, Q S G443 D N445 S, D S, D D R446 G, SG, S G, S T448 A A T449 A T450 I T451 A, S A, S R453 G, S, K G, S, KP454 S A455 V, T, AAAAPA AAAAPA AAAAPA T456 A, I T457 P G459 D S460 PS461 R P462 L, del L G463 V, D V, D P464 L, Q, PTHAAA PTHAAA PTHAAA T465I, S S467 T H468 L, R, Q L, R, Q L, R, Q G470 D C472 R G474 S G476 DY477 Y S478 Y P480 S S V482 A, I, T A, I, T A, I, T C483 R S485 T T G486S, D T488 I C489 R Q490 L V491 I L492 Q N493 D Y495 C Y496 F Q498 K C499G L500 I

The inventors of the present inventions surprisingly found thatintroduction of these modifications can yield polypeptides havingcellobiohydrolase activity with elevated IT50 values, which the skilledperson can learn from the examples below, particularly example 8.

Even more preferably, the variant of the polypeptides of the presentinvention as generally defined above comprises in a particularembodiment an amino acid sequence selected from the sequences withmutations with respect to SEQ ID NO: 2. optionally fused with aC-terminal 6×-His Tag, listed in the following Table 2.

TABLE 2 Mutations with respect to SEQ ID NO: 2: Consecutive NumberMutation Pattern with respect to Seq. ID NO: 2 1 G4C, A72C, Q349K 2 G4C,A72C, T344M, Q349K 3 G4C, A72C, T344M, D346G, Q349R 4 G4C, A72C, D320V,Q349K 5 G4C, A72C, P224L, F306Y, Q349R 6 G4C, A72C 7 A72V, D346A, T393A8 G4C, A72C, Q349R, R446S, T456A 9 G4C, W40R, A72C, T344M, Q349K 10A72V, D320V, D346A 11 G4C, A72C, N194Y, T243L, Q349R, Y374S, A375R 12G4C, A72C, Q349K, T448A, T449A 13 G4C, E65V, A72C, Y244H, Q349R 14 G4C,A72C, D202G, D320N, Q349R, A358E 15 G4C, A72C, D320V, Q349R 16 G4C,A72C, Q349K, S86T 17 A72V, T335I, D346A, T393A, P436S 18 G4C, A72C,E183V, K304R, Q349K 19 G4C, A72C, T243G, Q349R, Y374P, A375M 20 G4C,A72C, Q349R, T465I 21 G4C, A72C, Q349R 22 G4C, A72C, N194V, T243M,Q349R, Y374A, A375T 23 G4C, D64N, A72C, Q349R, A358E, P464Q 24 G4C,A72C, Q349K, Q28R, S193T, Q490L 25 G4C, A72C, E183K, Q349K 26 G4C, A72C,S311N, Q349K, A455T 27 G4C, A72C, N194K, Q349R, Y374P, A375Q 28 G4C,A72C, D181N, Q349K 29 W40R, D320V, Q349K, T393A, N445D 30 W40R, T335I,D346A, T393A 31 Q1L, G4C, A72C, D181N, E183K, N327Y, Q349R 32 A72C,L119L, T335I, Q349R, G486D 33 G4C, A72C, N194K, T243P, Q349R, Y374H,A375E 34 G4C, A72V, Q349R, P462del 35 G4C, A72C, S236Y, Q349R 36 G4C,A72C, S311G, Q349K 37 A72V, D320V, T335I, D346A, T393A, N445D 38 G4C,A72C, S86T, M234V, Q349K 39 Q1L, G4C, Q28R, E65V, A72C, K159KCGRNK,D181N, E183M, P224L, S311G, D320I, D346E, Q349K, T393V, Y422F, P442S,N445D, R446G, T457TAAATT, H468L, V482I 40 G4C, A72C, G251R, Q349R 41G4C, A72C, Q349K, D320V 42 A72V, T335I, D346A, T393A 43 G4C, A72C,E183K, Q349R 44 Q1L, G4C, A72C, H203R, Q349K, P442S 45 G4C, A72C, Q349K,G434S, G470D 46 G4C, W40R, A72C, Q349K 47 G4C, A72C, Q349R, V367A 48Q1L, G4C, A6V, C50S, A72C, I180N, D181N, E183K, Q349R, T457P, C472R,C499G 49 G4C, A72C, S311G, D320V, Q349K 50 W40R, T335I, D346A, T393A,P436S 51 Q1L, G4C, A72C, D181N, E183K, T243S, Q349R, P386S 52 A72V,D346A, T393A, N445D 53 Q1L, G4C, A72C, K154R, Q349K, T393I 54 G4C, A72C,N194G, T243F, Q349R, Y374P, A375R 55 A72V, D320V, D346A, T393A, N445D 56A72C, L119L, Q172Q, Q349K, T488I 57 G4C, A72C, E183V, Q349K 58 G4C,A72C, E183K, N318Y, Q349K 59 W40R, A221V, T449A, C483R 60 G4C, A72C,K92R, Q349K, N493D 61 Q1L, G4C, A72C, S90T, D181N, E183K, Q349R 62 G4C,A72C, Q349R, G459D 63 G4C, A72C, Q349R, Y422F 64 G4C, T48A, A72C, Q349R,P480S 65 E187K, D320V, P442del 66 G4C, S24N, E65K, A72C, Q349R, I430L,G439D 67 A72V, D320V, T335I, D346A, T393A, P436S 68 Q1L, G4C, A72C,S193P, Q349K, V482I 69 G4C, A72C, D320V, Q349K, G443D, L492Q 70 Q1L,G4C, A72C, DV152-K159, D181N, E183K, Q349R 71 Q1L, G4C, A72C, Q349K 72Q1L, G4C, A72C, D181N, E183K, M234L, V313I, Q349R, H468R 73 Q1L, G4C,A72C, D181N, E183K, I200N, Q349R 74 G4C, A72C, N194K, T243Y, Q349R,A375N 75 Q1L, G4C, Q28R, A72C, Q349K, H468L 76 G4C, E65V, A72C, Q349R 77D320V, Q349K 78 Q1L, G4C, A72C, S311G, Q349K, H468R 79 G4C, A72C, T243Q,Q349R, Y374P, A375M 80 Q1L, G4C, A72C, D320V, Q349R 81 Q1L, G4C, T15S,A72C, Y244F, Q349K 82 G4C, A72C, E183K, D346E, Q349R 83 Q1L, G4C, A72C,Q349K, T392M 84 G4C, A72C, D202N, S311N, Q349R, N493D 85 G4C, A72C,N194D, T243A, Q349R, Y374P, A375Y 86 G4C, A72C, N194Y, T243V, Q349R,Y374P 87 Q1L, G4C, A72C, Q349R 88 G4C, Q28R, E65K, A72C, S86T, D202N,H203R, S311N, D320I, A340G, D346A, Q349K, T393A, Y422F, P442S, R446S,H468L, V482A 89 G4C, A72C, D202N, Q349R 90 G4C, A72C, P224L, Q349R 91Q1L, G4C, A72C, D181N, E183K, T229M, A340T, Q349R, V491I 92 Q1L, G4C,A72C, D181N, Q349R 93 G4C, A72C, D320V, D346V, Q349K 94 Q1L, G4C, A72C,V152A, Q349K 95 Q1L, G4C, Q28R, A72C, Q349K 96 Q1L, G4C, A72C, Q349K,Y422F 97 G4C, A72C, D202V, D320V, Q349K 98 Q1L, G4C, A72C, Y155S, D181N,E183K, Q349R 99 Q1L, G4C, A72C, D181N, D247N, Q349K 100 Q1L, G4C, A68T,A72C, Q349K, G439D, R453S 101 Q1L, G4C, A72C, Q349K, H468R 102 Q1L, G4C,D64N, A72C, Q349K 103 G4C, A72C, E183K, Q349R, P464L 104 G4C, A72C,D181N, P224L, Q349K 105 G4C, A72C, N194I, T243Y, Q349R, Y374P, A375R 106Q1L, G4C, A72C, Q349K, P462L 107 Q1L, G4C, A72C, E183K, Q349R 108 G4C,A72C, S311G, Q349R 109 Q1L, G4C, A72C, S311N, Q349K, G463D 110 Q1L, G4C,A72C, S86T, Q349R 111 Q1L, G4C, A72C, D181N, E183K, G231D, Q349R 112Q1L, G4C, A72C, S89N, D181N, E183K, Q349R 113 Q1L, G4C, E65K, A72C,Q349K 114 Q1L, Q2P, G4C, W40R, E65M, A72C, S86T, S192L, D202N, H203R,S311D, D320I, T335I, D346G, Q349K, T392M, Y422F, R446G 115 Q1L, G4C,S24T, T26I, Q28R, N29Y, G30A, E65V, A68T, A72C, Y155C, D181N, E183M,Q190K, P224L, S311G, D320I, D346E, Q349K, T393V, Y422F, P442S, N445D,R446G, H468L, V482I 116 G4C, A72C, E183K, D202Y, N310D, Q349R 117 G4C,A72C, N194I, T243D, Q349R, Y374P, A375Y 118 Q1L, G4C, A72C, D181N,E183K, Q349R, T456I 119 G4C, Q28R, A72C, S86T, E183K, P224L, S311N,N318Y, T335I, D346G, Q349R, T393I, P441A, P442S, R446G, H468L, V482I 120Q1L, G4C, A72C, D181N, Q349K, T451S 121 Q1L, G4C, A72C, D181N, E183K,T243I, N246D, Q349R, T488I 122 Q1L, G4C, G23N, A72C, D110G, I116V,L119I, D181N, E183K, D211G, D293R, N310D, Q349R, Q362R, G363P, M364S 123G439V, N440E, P441S, P442Q 124 G431D, S431V, T433E, G434S, N435Q 125Q1L, G4C, Q28R, E65V, A68T, A72C, Y155C, D181N, E183M, P224L, S311G,D320I, D346E, Q349K, T393V, Y422F, P442S, N445D, R446G, H468L, V482I 126Q1L, G4C, A72C, S86T, D181N, E183K, Q349R, T393S 127 Q1L, G4C, A72C,D181N, E183K, Q349R, S485T 128 Q1L, G4C, G23N, A72C, V84A, D110G, D111H,I116E, F117Y, K118A, D181N, E183K, D293R, T295S, Q349R, M364L 129 Q1L,G4C, A72C, D181N, E183K, Q349R, R453K 130 Q1L, G4C, A72C, A145T, H203R,Q349K, T403K 131 Q1L, G4C, A72C, D181N, E183K, Q349R, N445S 132 Q1L,G4C, A72C, D181N, E183K, M234I, Q349R 133 Q1L, G4C, A72C, D181N, E183K,Q349R, T465S 134 Q1L, G4C, A72C, D181N, E183K, T297N, Q349R 135 Q1L,G4C, A72C, S311G, Q349K 136 G4C, Q28R, E65M, A72C, S86T, E183K, S192I,H203R, S311N, D346E, Q349K, T392M, T393A, Y422F, N445D, R446S 137 Q1L,G4C, A72C, D202N, Q349K, G486D 138 Q1L, G4C, A72C, S99T, D181N, E183K,Q349R, T450I 139 Q1L, G4C, A72C, I200F, Q349K, L500I 140 Q1L, G4C, A72C,D181N, E183K, Q349R, G434S 141 Q1L, G4C, A31S, A72C, D181N, E183K, Q349R142 Q1L, G4C, Q28L, A72C, D181N, E183K, Q349R 143 Q1L, G4C, A72C, D181N,E183K, Q349R, P436S 144 Q1L, G4C, A72C, D181N, E183K, T233S, Q349R 145Q1L, G4C, A72C, D202N, Q349K 146 Q1L, G4C, A68T, A72C, Q349K 147 Q1L,G4C, A21T, A72C, D181N, E183K, Q349R, P454S 148 Q1L, G4C, A72C, D346V,Q349K 149 Q1L, G4C, Y47F, A72C, D181N, E183K, Q349R, P436S, S461R 150Q1L, G4C, A72C, D181N, E183K, M234T, Q349R 151 Q1L, G4C, A72C, N157S,D181N, E183K, Q349R 152 Q1L, G4C, A72C, D181N, E183K, Q349R 153 G4C,A72C, N194Q, T243V, Q349R, Y374P, A375Y 154 Q1L, G4C, A72C, D181N,E183K, I314F, Q349R, N445D 155 Q1L, G4C, A72C, Q349K, T392K 156 Q1L,G4C, A72C, D181N, E183K, Q349R, T451A 157 Q1L, G4C, A72C, D181N, E183K,M234V, Q349R 158 Q1L, G4C, A21S, A72C, D181N, E183K, Q349R 159 Q1L, G4C,A72C, D181N, E183K, Q349R, N493D 160 Q1L, G4C, A72C, S311N, Q349K 161Q1L, G4C, Q28R, E65V, A72C, D181N, E183V, D228N, S311N, N318Y, D346E,Q349R, Y422F, P442S, N445D, R446G, H468L, V482T 162 G4C, A72C, N194C,Q349R, Y374C 163 Q1L, G4C, A72C, D181N, E183K, Q349R, A455V 164 Q1L,G4C, A72C, D181N, E183K, Q349R, T400S 165 Q1L, G4C, T26I, A72C, D181N,E183K, Q349R 166 Q1L, G4C, A72C, D181N, E183K, N310D, Q349R, T392S,G463D 167 Q1L, G4C, A72C, D129N, D181N, E183K, Q190L, G266S, I276V,Q349R, P386L, F427Y 168 Q1L, G4C, A72C, D181N, E183K, D202N, Q349R 169Q1L, G4C, A72C, Y155C, D181N, E183K, Q349R 170 Q1L, G4C, Q28R, E65V,A72C, D181N, E183M, P224L, S311G, D320I, D346E, Q349K, T393V, Y422F,P442S, N445D, R446G, P454PATAAA, H468L, V482I 171 Q1L, G4C, Q28R, E65V,A72C, K159KCGRNK, D181N, E183M, P224L, S311G, D320I, D346E, Q349K,T393V, Y422F, P442S, N445D, R446G, H468L, V482I 172 Q1L, G4C, A72C,D181N, E183K, N246K, Q349R 173 G4C, W40R, E65V, A72C, S86T, D181N,E183K, D202I, H203R, S311D, D320N, D346V, Q349R, T392M, T393A, Y422F,P442S, H468Q, V482A 174 Q1L, G4C, A72C, Y155C, Q349K 175 Q1L, G4C, A68T,A72C, D181N, E183K, Q349R 176 Q1L, G4C, S24T, T26I, Q28R, N29Y, G30A,Y47F, E65V, A68T, A72C, Y155C, D181N, E183M, Q190K, P224L, T229M, G231D,M234T, S311G, D320I, A340S, D346E, Q349K, D390E, T393V, Y422F, P442S,N445D, R446G, T448A, R453G, H468L, P480S, V482I 177 Q1L, G4C, W40R,E65M, A72C, S86T, S192L, D202N, H203R, S311D, D320I, T335I, D346G,Q349K, T392M, Y422F, R446G 178 Q1L, G4C, A72C, S86T, D181N, E183K,D320V, Q349R 179 G4C, Q28K, A72C, S86T, E183M, D202N, P224L, S311G,N318Y, D320N, D346A, Q349R, T392M, T393I, P442S, H468L, V482I 180 Q1L,Q2P, G4C, Q28R, W40R, E65K, A72C, D181N, S192L, D202I, H203R, P224L,S311G, D320I, D343A, D346A, Q349K, P442S, N445D, R446G, V482A 181 Q1L,G4C, Q28R, E65K, A72C, E183M, D202I, P224L, D320N, D346V, Q349K, T392M,T393V, Y422F, N445D, R446G, H468L, V482T 182 Q1L, G4C, Q28R, E65V, A72C,S86T, E183K, D202V, S311G, N318Y, D320I, D346G, Q349K, T393V, Y422F,N445D, R446S, H468Q, V482T 183 Q1L, G4C, W40R, E65M, A72C, D181N, E183K,S192P, D202N, P224L, S311D, N318Y, D320V, D346G, Q349K, T392M, N445D,R446G, H468L, V482T 184 Q1L, G4C, E65V, A72C, D181N, E183K, P224L,S311G, D320N, D346G, Q349R, T392M, T393I, R446G, H468L, V482I 185 Q1L,G4C, Q28R, E65V, A72C, G151GCGRSG, D181N, E183M, P224L, S311G, D320I,D346E, Q349K, T393V, Y422F, P442S, N445D, R446G, H468L, V482I 186 Q1L,G4C, E65M, A72C, D181N, E183M, D202Y, P224L, S311D, N318Y, D320I, T335I,D346A, Q349K, T392M, T393I, N445D, R446G, T448A, H468Q, V482A 187 Q1L,G4C, G23A, A72C, D110S, D111H, I116V, F117Y, K118A, L120M, D181N, E183K,D293H, G294A, N310E, Q349R, Q362G, M364S 188 Q1L, G4C, A72C, D181N,E183K, Q349R, T421I, G439D 189 G4C, Q28K, E65M, A72C, S86T, V152A,D181N, E183V, S192L, D202N, S311N, D320N, D346E, Q349R, T387A, T392M,T393I, Y422F, P442S, R446S, H468L, G476D, V482I 190 Q1L, G4C, W40R,E65V, A72C, S86T, E183V, G188C, S192T, D202Y, H203R, P224L, S311N,D320V, D346E, Q349K, T393V, Y422F, N445D, R446G, H468Q, V482I 191 Q1L,G4C, E65M, A72C, S86T, E183M, D202N, P224L, T335I, D346G, Q349K, T392M,T393A, P442S, N445D, R446G, H468Q, V482A 192 Q1L, G4C, Q28R, E65V, A72C,D181N, E183M, P224L, S311G, D320I, A340S, D346E, Q349K, D390E, T393V,Y422F, P442S, N445D, R446G, H468L, P480S, V482I 193 G4C, W40R, A72C 194Q1L, G4C, W40R, E65K, A72C, S86T, E183K, S192L, D202Y, P224L, D320I,D346E, Q349R, P442S, R446G, H468R, V482T 195 Q1L, G4C, A21T, T26I, Q28R,E65V, A68T, A72C, Y155C, D181N, E183M, Q190K, D202N, P224L, S311G,N318Y, D320I, D346E, Q349K, T393V, Y422F, N445D, R446G, R453G, H468Q,P480S, V482I 196 Q1L, G4C, Q28R, E65V, A72C, S86T, D181N, D202N, P224L,S311G, N318Y, D320I, T335I, D346E, Q349K, T393V, Y422F, N445D, R446G,H468Q, V482I 197 Q1L, G4C, Q28R, E65V, A72C, D181N, E183M, P224L, S311G,D320I, D346E, Q349K, T393V, Y422F, P442S, N445D, R446G, P464PTHAAA,H468L, V482I 198 Q1L, G4C, Q28R, E65V, A72C, E183M, S311G, D346E, Q349K,T393V, Y422F, P442S, N445D, R446G, H468L, V482I 199 Q1L, G4C, T7Q, A8S,N10T, S24T, T27Q, Q28R, N29T, V41T, G46S, Y47S, T52D, D57S, D64N, E65V,Q69K, A72C, D181N, P224L, S311D, N318Y, D320N, D346A, Q349K, T392M,T393I, Y422F, N445D, R446G, H468Q, V482I 200 Q1L, G4C, Q28R, E65V, A72C,K159KCGRNK, E183M, P224L, S311G, D320I, D346E, Q349K, T393V, Y422F,P442S, N445D, R446G, T457TAAATT, H468L, V482I 201 Q1L, G4C, Q28K, W40R,E65V, A72C, D181N, E183V, S192P, D202V, H203R, S311G, D320N, D346E,Q349K, T392M, T393A, Y422F, V482I 202 Q1L, G4C, Q28R, E65V, A72C, G139S,D181N, E183M, P224L, S311G, D320I, D346E, Q349K, T393V, Y422F, P442S,N445D, R446G, H468L, V482I 203 Q1L, G4C, Q28R, E65V, A72C, S86T, D181N,E183M, D202N, P224L, S311G, N318Y, T335I, D346E, Q349K, T393V, Y422F,N445D, R446G, H468L, V482I 204 Q1L, G4C, Q28R, E65K, A72C, D181N, S311N,N318Y, D346E, Q349K, T393V, Y422F, N445D, R446G, H468Q, V482I 205 Q1L,G4C, W40R, A72C, S192L, D202N, H203R, P224L, S311N, D320I, T335I, D346V,Q349R, T393I, N445D, R446G, H468Q, V482T 206 Q1L, G4C, Q28K, E65K, A72C,E183M, D202N, P224L, T229S, S311G, D320I, T335I, D346V, Q349R, T393V,H468Q, V482A 207 Q1L, G4C, Q28R, G30A, E65M, A72C, D181N, D202N, P224L,S311D, N318Y, D346E, Q349K, T392M, T393V, Y422F, P442S, N445D, R446S 208Q1L, G4C, Q28R, E65V, A72C, S68T, E183M, S311N, T335I, D346E, Q349K,T393V, Y422F, N445D, R446G, H468L, V482T 209 Q1L, G4C, Q28K, E65K, A72C,D181N, D202N, S311N, N318Y, T335I, D346E, Q349K, T393V, Y422F, P442S,N445D, R446G, H468L, V482I 210 T243C, A375C, N194C, Y374C 211 Q1L, G4C,Q28K, E65K, A72C, S86T, E183M, S311G, D346V, Q349R, R392M, T393V, Y422F,P442S, N445D, R446R, H468L, S478Y, V482I 212 Q1L, G4C, Q28R, E65V, A72C,D181N, E183M, S311G, D346E, Q349K, T393V, Y422F, P442S, N445D, R446G,H468L, V482I 213 Q1L, G4C, Q28R, E65V, A72C, S86T, D181N, D202N, S311N,N318Y, D320I, T335I, D346E, Q349K, T393I, Y422F, N445D, R446G, H468Q,V482I 214 Q1L, G4C, Q28R, E65K, A72C, S86T, E183K, D202N, P224L, S311G,T335I, D346A, Q349K, T393V, Y422F, N445D, R446G, H468L, V482T 215 Q1L,G4C, Q28R, E65V, A72C, L120P, D181N, E183M, P224L, S311G, D320I, D346E,Q349K, T393V, Y422F, P442S, N445D, R446G, H468L, V482I 216 Q1L, G4C,Q28K, E65V, A72C, S86T, E183M, S311N, N318H, D320V, D346V, Q349K, T392M,T393A, R446S, H468L, V482I 217 Q1L, G4C, Q28K, E65V, A72C, D202N, H203R,S311G, T335I, D346V, Q349K, T393A, P442S, R446G, H468L, V482I 218 Q1L,G4C, Q28R, E65V, A72C, E183M, S311G, D320I, D346E, Q349K, T393V, Y422F,P442S, N445D, R446G, H468L, V482I 219 Q1L, G4C, Q28R, E65K, A72C, D181N,D202N, H203R, P224L, S311D, D346G, Q349K, P442S, R446S, H468Q, V482T 220Q1L, G4C, Q28R, E65M, A72C, D181N, E183M, S311G, N318Y, D320I, T335I,D346E, Q349K, D390E, T393V, Y422F, P442S, N445D, R446G, H468Q, V482T 221Q1L, G4C, T27S, Q28R, E65V, Q69R, A72C, L120P, D181N, E183M, D202N,D247N, S311G, D346E, Q349K, K355Q, T387S, T393V, Y422F, N445D, R446G,T451S, G463V, H468Q, V482T, S485T 222 Q1L, G4C, Q28R, E65K, A72C, E183V,S192T, D202N, S311G, D320V, D346A, Q349K, P442S, N445D, R446G, H468R,V482A 223 Q1L, G4C, W40R, E65V, A72C, S86T, E183M, D202N, P224L, S311G,D320V, D346E, Q349R, T393I, P442S, R446G, H468R, V482I 224 Q1L, G4C,Q28N, E65K, A72C, D181N, E183M, D202N, H203R, S311G, N318Y, D320N,Q349R, T393V, Y422F, P442S, N445D, R446G, H468L, V482I 225 Q1L, G4C,Q28R, V41T, G46S, Y47S, T52D, E65V, A72C, D181N, 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Q28R, E65V, A72C,D181N, E183M, P224L, S311G, D320I, D346E, Q349K, T393V, Y422F, P442S,N445D, R446G, P454PVRPQP, H468L, V482I 380 Q1L, G4C, Q28R, A72C, D181N,E183V, S192M, D202N, P224L, S311D, N318Y, D320N, D346E, Q349R, T393V,Y422F, P442S, R446G, H468L, P480S 381 Q1L, G4C, Q28R, A72C, S86T, D181N,S192L, D202N, P224L, S311G, N318Y, D320V, T335I, D345E, D346A, Q349K,Y422F, P442S, N445D, R446G, H468Q, H505Q 382 Q1L, G4C, Q28R, E65V, A72C,S86T, D181N, E183K, P224L, S311G, N318Y, D320I, D346A, Q349K, T393V,Y422F, P442S, N445D, R446G, H468L, V482I 383 Q1L, G4C, Q28R, G30A, E65V,A68T, A72C, D181N, E183M, D202N, P224L, D346E, Q349K, T393V, Y422F,N445D, R446G, T448A, R453G, H468Q, V482I 384 Q1L, G4C, Q28R, E65V, A72C,K92R, L120P, D181N, E183M, D202N, S236F, T280A, S311G, D346E, Q349K,K355Q, T387S, T393V, P402S, D404N, Y422F, N445D, R446G, T451S, G463V,H468Q, V482I, S485T 385 Q1L, G4C, Q28R, E65V, A72C, D181N, D202N, S236F,S311G, T335I, D346E, Q349K, T387S, T393V, Y422F, N445D, R446G, G463V,H468Q, V482I, S485T 386 Q1L, G4C, Q28R, E65M, A72C, D181N, E183K, D202N,P224L, S311G, D320I, D346E, Q349K, T393V, Y422F, P442S, N445D, R446G,H468Q, V482I 387 Q1L, G4C, A21T, T26I, Q28R, G30A, E65V, A68T, A72C,Y155C, D181N, E183M, Q190K, D202N, S311G, D320I, D346E, Q349K, T393V,Y422F, N445D, R446G, T448A, H468Q, V482I 388 Q1L, G4C, Q28R, E65V, A72C,D181N, D202N, S311G, T335I, D346A, Q349K, T393I, Y422F, P442S, N445D,R446G, H468L, V482I 389 Q1L, G4C, Q28R, E65V, A72C, D181N, E183M, P224L,D247N, S311G, D320I, D346E, Q349K, T387S, T393V, Y422F, P442S, N445D,R446G, H468L, V482I 390 Q1L, G4C, Q28R, E65V, A72C, D181N, D202N, P224L,S311G, D346A, Q349K, T393V, Y422F, N445D, R446G, H468L, V482I 391 Q1L,G4C, Q28R, E65V, A72C, K92R, K118Q, D181N, E183M, D202N, S236F, S311N,N318I, D346E, Q349K, K355Q, T387S, T393V, D404N, Y422F, N445D, R446G,H468Q, V482I 392 Q1L, G4C, T27S, Q28R, E65V, Q69R, A72C, K118Q, D181N,E183M, D202N, D247N, I277V, S311G, T335I, D346E, Q349K, K355Q, T393V,Y422F, N445D, R446G, H468Q, V482T 393 Q1L, G4C, Q28R, G30A, E65V, A72C,Y155C, D181N, E183M, D202N, M234T, S311G, D346E, Q349K, T393V, Y422F,N445D, R446G, T448A, R453G, H468Q, V482T 394 Q1L, G4C, Q28R, E65V, A72C,D181N, E183M, P224L, S311G, D320I, D346E, Q349K, T393V, Y422F,G434GAAATG, P442S, N445D, R446G, H468L, V482I 395 Q1L, G4C, Q28R, E65V,Q69R, A72C, K92R, D181N, D202N, D247N, I277V, S311G, N318D, T335I,D346E, Q349K, K355Q, T387S, T393V, Y422F, N445D, R446G, G463V, H468Q,V482I 396 Q1L, G4C, T27S, Q28R, E65V, Q69R, A72C, D181N, D202N, S236F,I277V, S311G, T335I, D346E, Q349K, K355Q, T387S, T393V, Y422F, N445D,R446G, G463V, H468Q, V482I 397 Q1L, G4C, N10D, Q28R, E65V, Q69R, A72C,K92R, K118Q, D181N, E183M, D202N, S311G, T335I, D346E, Q349K, T387S,T393V, Y422F, N445D, R446G, P462L, H468Q, V482T, S485T 398 Q1L, G4C,A21T, Q28R, G30A, E65V, A72C, Y155C, D181N, E183M, D202N, M234T, S311G,T335I, D346E, Q349K, T393V, Y422F, N445D, R446G, R453G, H468Q, V482T 399Q1L, G4C, Q28R, E65V, A72C, D181N, E183M, P224L, S311G, D320I, D346E,Q349K, T393V, Y422F, P442S, N445D, R446G, T457TAAATT, H468L, V482I 400Q1L, G4C, T26I, Q28R, E65V, A68T, A72C, Y155C, D181N, Q190K, D202N,P224L, M234T, S311G, D320I, D346E, Q349K, D390E, T393V, Y422F, N445D,R446G, R453G, H468Q, V482I 401 Q1L, G4C, Q28K, E65V, A72C, S86T, E183K,S192L, D202N, D320N, T335I, D346A, Q349R, T393A, Y422F, P442S, R446G,H468R, V482A 402 Q1L, G4C, Q28R, E65V, A72C, D181N, E183M, P224L, S236F,S311G, D320I, D346E, Q349K, T393V, Y422F, P442S, N445D, R446G, H468L,V482I 403 Q1L, G4C, T26I, Q28R, E65V, A68T, A72C, Y155C, D181N, D202N,M234T, S311G, T335I, D346E, Q349K, T393V, Y422F, N445D, R446G, H468Q,V482T 404 Q1L, G4C, A21T, T26I, Q28R, N29Y, E65V, A72C, Y155C, D181N,E183M, D202N, P224L, M234T, S311G, D320I, T335I, A340S, D346E, Q349K,D390E, T393V, Y422F, N445D, R446G, T448A, R453G, H468Q, P480S, V482I 405Q1L, G4C, Q28R, E65K, A72C, D202I, P224L, S311G, N318Y, D320V, T335I,D346A, Q349R, T392M, T393A, N445D, R446G, H468L 406 Q1L, G4C, E65M,A72C, S86T, E183V, D202I, P224L, S311G, N318Y, D320V, D346A, Q349K,P442S, N445D, R446G, H468L, V482A 407 Q1L, G4C, N10D, Q28R, E65V, A72C,K92R, D181N, E183M, D202N, S311G, D346E, Q349K, T387S, T393V, Y422F,N445D, R446G, T451S, P462L, H468Q, V482T 408 Q2S, G4C, A6L, T7Q, A8S,N10T, Q28K, E65M, A72C, S86T, E183M, D202N, P224L, S311G, D320I, T335I,(DeletionS437-P441), D346E, Q349K, T393V, Y422F, N445D, R446G, H468Q,V482T 409 Q2S, G4C, A6L, T7Q, A8S, N10T, S24T, T26I, Q28R, N29Y, G30A,E65V, A72C, E183M, P224L, S311G, D320I, D346E, Q349K, T393V, Y422F,P442S, N445D, R446G, H468L, V482I 410 Q1L, G4C, Q28R, E65V, A72C, K92R,D181N, E183M, P224L, I277V, S311G, D320I, D346E, Q349K, T393V, Y422F,P442S, N445D, R446G, H468L, V482I 411 Q2S, G4C, A6L, T7Q, A8S, N10T,Q28R, E65V, A72C, E183M, P224L, S311G, D320I, D346E, Q349K, T393V,Y422F, P442S, N445D, R446G, H468L, V482I 412 Q2S, G4C, A6L, T7Q, A8S,N10T, Q28R, E65V, A72C, E183M, P224L, S311G, D320I, D346E, Q349K, T393V,Y422F, P442S, N445D, R446G, P454PVRPQP, H468L, V482I 413 Q2S, G4C, A6L,T7Q, A8S, N10T, Q28R, E65V, A72C, E183M, P224L, S311G, D320I, D346E,Q349K, T393V, Y422F, P442S, N445D, R446G, P464THAAA, H468L, V482I 414Q2S, G4C, A6L, T7Q, A8S, N10T, Q28R, E65V, A72C, E183M, P224L, S311G,D320I, D346E, Q349K, T393V, Y422F, P442S, N445D, R446G, P464PTHAAA,H468L, V482I 415 Q2S, G4C, A6L, T7Q, A8S, N10T, Q28R, E65V, A72C,G151GCGRSG, E183M, P224L, S311G, D320I, D346E, Q349K, T393V, Y422F,P442S, N445D, R446G, H468L, V482I 416 Q2S, G4C, A6L, T7Q, A8S, N10T,Q28R, E65V, A72C, K159KCGRNK, E183M, P224L, S311G, D320I, D346E, Q349K,T393V, Y422F, P442S, N445D, R446G, H468L, V482I 417 Q2S, G4C, A6L, T7Q,A8S, N10T, Q28R, E65V, A72C, E183M, P224L, S311G, D320I, D346E, Q349K,T393V, Y422F, P442S, N445D, R446G, P454ATAAA, H468L, V482I 418 Q2S, G4C,A6L, T7Q, A8S, N10T, Q28K, E65K, A72C, E183M, S311G, N318Y, D320I,T335I, D346E, Q349K, T393V, Y422F, N445D, R446G, H468Q, V482T 419 Q2S,G4C, A6L, T7Q, A8S, N10T, Q28K, E65V, A72C, E183M, D202N, P224L, S311G,T335I, D346E, Q349K, T393V, Y422F, P442S, N445D, R446G, H468Q, V482I 420Q2S, G4C, A6L, T7Q, A8S, N10T, Q28R, E65M, A72C, S86T, E183M, S311N,N318Y, T335I, D346E, Q349K, T393V, Y422F, N445D, R446G, H468Q, V482I 421Q2S, G4C, A6L, T7Q, A8S, N10T, Q28R, E65M, A72C, E183M, S311G, N318Y,D346E, Q349K, T393V, Y422F, P442S, N445D, R446G, H468Q, V482I 422 Q2S,G4C, A6L, T7Q, A8S, N10T, Q28R, E65V, A72C, E183M, D202N, P224L, S311G,N318Y, T335I, D346E, Q349K, T393V, Y422F, P442S, N445D, R446G, H468Q,V482I 423 Q2S, G4C, A6L, T7Q, A8S, N10T, Q28R, E65V, A72C, E183M, P224L,S311G, N318Y, T335I, D346A, Q349K, T393V, Y422F, P442S, N445D, R446G,H468L, V482T 424 Q2S, G4C, A6L, T7Q, A8S, N10T, Q28R, E65V, A72C, P224L,S311D, N318Y, D320N, D346A, Q349K, T392M, T393I, Y422F, N445D, R446G,H468Q, V482I 425 Q2S, G4C, A6L, T7Q, A8S, N10T, Q28R, E65K, A72C, S311N,N318Y, D346E, Q349K, T393V, Y422F, (DeletionbeiG439-G444), N445D, R446G,H468Q, V482I 426 Q2S, G4C, A6L, T7Q, A8S, N10T, Q28K, N29Y, E65K, A72C,S311G, T335I, D346E, Q349K, T393V, Y422F, N445D, R446G, H468Q, V482T 427Q2S, G4C, A6L, T7Q, A8S, N10T, S24T, T26I, Q28R, N29Y, G30A, E65V, A68T,A72C, E183M, Q190K, P224L, S311G, D320I, D346E, Q349K, T393V, Y422F,P442S, N445D, R446G, H468L, V482I 428 Q2S, G4C, A6L, T7Q, A8S, N10T,S24T, T26I, Q28R, N29Y, G30A, Y47F, E65V, A68T, A72C, E183M, Q190K,P224L, T229M, G231D, M234T, S311G, D320I, A340S, D346E, Q349K, D390E,T393V, N445D, R446G, T448A, R453G, H468L, P480S, V482I, Y422F, P442S 429Q2S, G4C, A6L, T7Q, A8S, N10T, Q28R, E65V, A72C, K159KCGRNK, E183M,P224L, S311G, D320I, D346E, Q349K, T393V, Y422F, P442S, N445D, R446G,T457TAAATT, H468L, V482I 430 Q2S, G4C, A6L, T7Q, A8S, N10T, Q28R, E65V,A72C, E183M, P224L, S311G, D320I, D346E, Q349K, T393V, Y422F, P442S,N445D, R446G, T457TAAATT, H468L, V482I 431 Q1L, G4C, A72C, S86T, D181N,E283K, D202V, P224L, S311G, D320V, D346E, Q349R, T393A, Y422F, P442S,N445D, R446G, H468L 432 Q1L, G4C, Q28R, E65V, A72C, K159CGRNKE183M,P224L, S311G, D320I, D346E, Q349K, T393V, Y422F, P442S, N445D, R446G,H468L, V482I 433 Q2S, G4C, A6L, T7Q, A8S, N10T, S24T, Q28R, E65K, A72C,D202N, S311G, T335I, D346E, Q349K, T393I, Y422F, N445D, R446G, H468Q,V482T 434 Q2S, G4C, A6L, T7Q, A8S, N10T, Q28R, E65M, A72C, E183K, D202N,P224L, S311G, D320I, D346E, Q349K, T393V, Y422F, P442S, N445D, R446G,H468Q, V482I 435 Q1L, G4C, Q28K, E65M, A72C, S86T, E183M, D202N, P224L,S311G, D320I, T335I, D346E, Q349K, T393V, Y422F, (S437-P441), N445D,R446G, H468Q, V482T 436 Q1L, G4C, S24T, T26I, Q28R, N29Y, G30A, E65V,A72C, E183M, P224L, S311G, D320I, D346E, Q349K, T393V, Y422F, P442S,N445D, R446G, H468L, V482I 437 Q1L, G4C, Q28R, E65V, A72C, E183M, P224L,S311G, D320I, D346E, Q349K, T393V, Y422F, P442S, N445D, R446G,T457TAAATT, H468L, V482I 438 Q1L, G4C, Q28R, E65V, A72C, E183M, P224L,S311G, D320I, D346E, Q349K, T393V, Y422F, P442S, N445D, R446G,P454PVRPQP, H468L, V482I 439 Q1L, G4C, Q28R, E65V, A72C, E183M, P224L,S311G, D320I, D346E, Q349K, T393V, Y422F, P442S, N445D, R446G,P464THAAA, H468L, V482I 440 Q1L, G4C, Q28R, E65V, A72C, E183M, P224L,S311G, D320I, D346E, Q349K, T393V, Y422F, P442S, N445D, R446G,P454PATAAA, H468L, V482I 441 Q1L, G4C, Q28R, E65V, A72C, E183M, P224L,S311G, D320I, D346E, Q349K, T393V, Y422F, G434GAAATG, P442S, N445D,R446G, H468L, V482I 442 Q1L, G4C, Q28R, E65V, A72C, D181N, E183M, P224L,S311G, D320I, D346E, Q349K, T393V, Y422F, P442S, N445D, R446G, H468L,V482I, Q1L, G4C, Q28R, E65V, A72C, E183M, P224L, S311G, D320I, D346E,Q349K, T393V, Y422F, P442S, N445D, R446G, H468L, V482I 443 Q1L, G4C,Q28R, E65V, A72C, G151GCGRSG, E183M, P224L, S311G, D320I, D346E, Q349K,T393V, Y422F, P442S, N445D, R446G, H468L, V482I 444 Q1L, G4C, Q28R,E65V, A72C, E183M, P224L, S311G, D320I, D346E, Q349K, T393V, Y422F,P442S, N445D, R446G, P454ATAAA, H468L, V482I 445 Q1L, G4C, Q28R, E65K,A72C, S311N, N318Y, D346E, Q349K, T393V, Y422F, (DG439-G444) N445D,R446G, H468Q, V482I 446 Q1L, G4C, S24T, T26I, Q28R, N29Y, G30A, E65V,A68T, A72C, E183M, Q190K, P224L, S311G, D320I, D346E, Q349K, T393V,Y422F, P442S, N445D, R446G, H468L, V482I 447 Q1L, G4C, S24T, T26I, Q28R,N29Y, G30A, Y47F, E65V, A68T, A72C, E183M, Q190K, P224L, T229M, G231D,M234T, S311G, D320I, A340S, D346E, Q349K, D390E, T393V, N445D, R446G,T448A, R453G, H468L, P480S, V482I, Y422F, P442S 448 Q1L, G4C, A21T,T26I, Q28R, N29Y, G30A, E65V, A68T, A72C, D181N, Q190K, D202N, T229M,M234T, S311G, D320I, A340S, D346E, Q349K, D390E, T393V, Y422F, N445D,R446G, T448A, H468Q, V482I 449 Q1L, G4C, N10D, T27S, Q28R, E65V, A72C,K92R, K118Q, D181N, D202N, S236F, I277V, S311G, D346E, Q349K, K355Q,T387S, T393V, D404N, N417Y, Y422F, N445D, R446G, P462L, H468Q, V482I,S485T 450 Q1L, G4C, A21T, T26I, Q28R, G30A, E65V, A68T, A72C, Y155C,D181N, Q190K, D202N, P224L, T229M, S311N, D320I, D346E, Q349K, T393V,Y422F, N445D, R446G, R453G, H468Q, V482I 451 Q1L, G4C, N10D, T27S, Q28R,E65V, A72C, K92R, K118Q, D181N, E183M, D202N, D247N, S311G, D320I,T335I, D346E, Q349K, T393V, Y422F, N445D, R446G, H468Q, V482T 452 Q1L,G4C, T26I, Q28R, G30A, E65V, A72C, Y155C, D181N, D202N, T229M, G231D,S311G, T335I, D346E, Q349K, T393V, Y422F, N445D, R446G, T448A, R453G,H468Q, V482T 453 Q1L, G4C, S24R, 726I, Q28R, G30A, E65V, A72C, D181N,E183M, D202N, P224L, S311N, T335I, D346E, Q349K, D390E, T393V, Y422F,N445D, R446G, H468Q, V482T 454 Q1L, G4C, N10D, T27S, Q28R, E65V, Q69R,A72C, D181N, E183M, D202N, S236F, D247N, I277V, S311N, T335I, D346E,Q349K, T387S, T393V, N417Y, Y422F, T433S, N445D, R446G, P462L, H468Q,V482I 455 Q1L, G4C, A21T, Q28R, E65V, A72C, Y155C, D181N, D202N, P224L,S311G, N318Y, D346E, Q349K, D390E, T393V, Y422F, N445D, R446G, T448A,H468Q, V482I 456 Q1L, G4C, T26I, Q28R, N29Y, E65V, A68T, A72C, Y155C,D181N, E183M, D202N, P224L, M234T, S311N, N318Y, D346E, Q349K, T393V,Y422F, N445D, R446G, T448A, R453G, H468Q, V482I 457 Q1L, G4C, A21T,T26I, Q28R, N29Y, E65V, A68T, A72C, Y155C, D181N, D202N, P224L, T229M,M234T, S311G, N318Y, D346E, Q349K, T393V, Y422F, N445D, R446G, R453G,H468Q, V482I 458 Q1L, G4C, N10D, Q28R, E65V, A72C, K92R, K118Q, D181N,E183M, D202N, P224L, S236F, D247N, F306L, S311G, D346E, Q349K, K355Q,T393V, D404N, N417Y, Y422F, N445D, R446G, H468Q, V482I, S485T 459 Q1L,G4C, N10D, Q28R, E65V, A72C, K92R, K118Q, D181N, E183M, D202N, S236F,D247N, S311G, N318I, T335I, D346E, Q349K, K355Q, T393V, Y422F, T433S,N445D, R446G, T451S, G463V, H468Q, V482I 460 G4C, Q28K, E65M, A72C,S86T, G151GCGRSG, E183M, D202N, P224L, S311G, T335I, D346E, Q349K,T393V, Y422F, P442S, N445D, R446G, H468L, V482I 461 G4C, Q28K, E65M,A72C, S86T, K159KCGRNK, E183M, D202N, P224L, S311G, T335I, D346E, Q349K,T393V, Y422F, P442S, N445D, R446G, H468L, V482I 462 G4C, Q28K, E65M,A72C, S86T, G151GCGRSG, K159KCGRNK, E183M, D202N, P224L, S311G, T335I,D346E, Q349K, T393V, Y422F, P442S, N445D, R446G, H468L, V482I 463 G4C,Q28K, E65M, A72C, S86T, G151GCGRSG, E183M, D202N, P224L, S311G, T335I,D346E, Q349K, T393V, Y422F, P442S, N445D, R446G, T457TAAATT, H468L,V482I 464 G4C, Q28K, E65M, A72C, S86T, K159KCGRNK, E183M, D202N, P224L,S311G, T335I, D346E, Q349K, T393V, Y422F, P442S, N445D, R446G,T457TAAATT, H468L, V482I 465 G4C, Q28K, E65M, A72C, S86T, G151GCGRSG,E183M, D202N, P224L, S311G, T335I, D346E, Q349K, T393V, Y422F,G434GAAATG, P442S, N445D, R446G, T457TAAATT, H468L, V482I 466 G4C, Q28K,E65M, A72C, S86T, K159KCGRNK, E183M, D202N, P224L, S311G, T335I, D346E,Q349K, T393V, Y422F, G434GAAATG, P442S, N445D, R446G, T457TAAATT, H468L,V482I 467 G4C, Q28K, E65M, A72C, S86T, G151GCGRSG, K159KCGRNK, E183M,D202N, P224L, S311G, T335I, D346E, Q349K, T393V, Y422F, G434GAAATG,P442S, N445D, R446G, H468L, V482I, T457TAAATT, 468 Q1L, G4C, Q28K, E65V,A72C, D181N, E183M, D202N, P224L, S311G, T335I, D346E, Q349K, T393V,Y422F, P442S, N445D, R446G, H468Q, V482I 469 Q1L, G4C, Q28K, E65V, A72C,S86T, D181N, E183K, S192L, D202V, S311G, D320I, D346V, Q349R, T393A,Y422F, P442S, N445D, R446G, H468Q, V482I 470 Q1L, G4C, Q28R, E65V, A72C,S86T, D181N, D202N, P224L, S311N, N318Y, D320I, D346A, Q349K, T393V,Y422F, N445D, R446G, H468L, V482T 471 Q1L, G4C, A21T, T26I, Q28R, G30A,E65V, A68T, A72C, D181N, E183M, D202N, T229M, S311G, A340S, D346E,Q349K, T393V, Y422F, N445D, R446G, H468Q, V482T

In a further aspect, the present invention discloses a nucleic acidencoding the polypeptide of the present invention. The nucleic acid is apolynucleotide sequence (DNA or RNA) which is, when set under control ofan appropriate promoter and transferred into a suitable biological hostor chemical environment, processed to the encoded polypeptide, wherebythe process also includes all post-translational andpost-transcriptional steps necessary. The coding sequence can be easilyadapted by variation of degenerated base-triplets, alteration of signalsequences, or by introduction of introns, without affecting themolecular properties of the encoded protein. The nucleic acid of thepresent invention has preferably at least 95%, more preferably at least97%, and most preferably 100% identity to SEQ ID NO: 1. The presentinvention also provides a vector comprising this nucleic acid and a hosttransformed with said vector.

The present invention also discloses methods for the production ofpolypeptides of the present invention and variants thereof in varioushost cells, including yeast and fungal hosts. It also discloses the useof the resulting strains for the improvement of protein properties byvariation of the sequence. Furthermore, the present invention disclosesmethods for the application of such polypeptides in the hydrolysis ofcellulose.

A further aspect of the invention discloses vectors and methods for theproduction of protein variants of SEQ ID NO; 2. expressing them in yeastand testing their activity on cellulosic material by measuring thereleased mono- and/or oligomeric sugar molecules.

The present invention further relates to a method of producing acellobiohydrolase protein, comprising the steps:

-   -   a. obtaining a host cell, which has been transformed with a        vector comprising the nucleic acid of the present invention;    -   b. cultivation of the host cell under conditions under which the        cellobiohydrolase protein is expressed; and    -   c. recovery of the cellobiohydrolase protein.

In a particular embodiment, this method of producing a cellobiohydrolaseprotein is restricted to a method for the production of acellobiohydrolase protein as provided by this invention, such as havingthe IT50 value given above, and/or being one of the specific variants ofSEQ ID NO2 or SEQ ID NO: 5 as provided with this application anddescribed in detail elsewhere in this specification.

In a more preferred embodiment, the host cell is derived from the groupconsisting of Saccharomyces, Schizosaccharomyces, Kluyveromyces, Pichia,Hansenula, Aspergillus, Trichoderma, Penicillium, Candida and Yarrowina.The host cell is preferably capable of producing ethanol, wherein mostpreferred yeasts include Saccharomyces cerevisiae, Pichia stipitis,Pachysolen tannophilus. or a methylotrophic yeast, preferably derivedfrom the group of host cells comprising Pichia methanolica, Pichiapastoris, Pichia angusta, Hansenula polymorpha.

It has surprisingly been found that the polypeptide according to thepresent invention and variants thereof can be expressed from yeast athigh levels. “Yeast” shall herein refer to all lower eukaryoticorganisms showing a unicellular vegetative state in their life cycle.This especially includes organisms of the class Saccharomycetes, inparticular of the genus Saccharomyces, Pachysolen, Pichia, Candida,Yarrowina, Debaromyces, Klyveromyces, Zygosaccharomyces.

Thus, one aspect of the invention relates to the expression of theclaimed polypeptide and variants thereof in yeast. The efficientexpression of this fusion protein (SEQ ID NO: 2) and derivative proteinvariants of SEQ ID NO: 2 from yeast can be achieved by insertion of thenucleic acid molecule of SEQ ID NO: 1 starting from nucleotide position1 into an expression vector under control of at least one appropriatepromoter sequence and fusion of the nucleotide molecule to anappropriate signal peptide, for example to the signal peptide of themating factor alpha of Saccharomyces cerevisiae.

In a preferred embodiment, the polypeptide of the present invention andvariants thereof are expressed and secreted at a level of more than 100mg/l, more preferably of more than 200 mg/l, particularly preferably ofmore than 500 mg/l, or most preferably of more than 1 g/l into thesupernatant after introduction of a nucleic acid encoding a polypeptidehaving an amino acid sequence with at least 85% sequence identity to theSEQ ID NO: 2 into a yeast. To determine the level of expression inyeast, the cultivation and isolation of the supernatant can be carriedout as described in Example 3.

A further aspect the invention discloses methods for the production of apolypeptide according to the present invention in a filamentous fungus,preferably in a fungus of the genus Aspergillus or Trichoderma. morepreferably in a fungus of the genus Trichoderma, most preferably inTrichoderma reesei. “Filamentous fungi” or “fungi” shall herein refer toall lower eukaryotic organisms showing hyphal growth during at least onestate in their life cycle. This especially includes organisms of thephylum Ascomycota and Basidiomycota, in particular of the genusTrichoderma, Talaromyces, Aspergillus, Penicillium, Chrysosporium,Phanerochaete, Thermoascus, Agaricus, Pleutrus, Irpex. The polypeptideis expressed by fusion of the coding region of a compatible signalsequence to the nucleic acid molecule starting with nucleotide position52 of SEQ ID NO: 3. as it was done in SEQ ID NO: 3 with the signalsequence of the Trichoderma reesei CBHI, and the positioning of thefusion peptide under control of a sufficiently strong promoter followedby transfer of the genetic construct to the host cell. Examples for suchpromoters and signal sequences as well as techniques for an efficienttransfer have been described in the art.

In a further aspect the present invention further relates to a methodfor identifying a polypeptide or polypeptides having cellobiohydrolaseactivity, comprising the steps of:

-   -   a. Generating a library of mutant genes encoding mutant proteins        by mutagenesis of a nucleic acid according to claim 9 or a        nucleic acid having the sequence defined by SEQ ID NO: 6        (encoding SEQ ID NO: 5), preferably having the sequence defined        by SEQ ID NO: 1;    -   b. Inserting each mutant gene into an expression vector;    -   c. Transforming yeast cells with each expression vector to        provide a library of yeast transformants;    -   d. Cultivation of each yeast transformant under conditions under        which the mutant protein is expressed and secreted;    -   e. Incubating the expressed mutant protein with a substrate;    -   f. Determining the catalytic activity of the mutant protein;    -   g. Selecting a mutant protein according to the determined        catalytic activity.

Specifically, step d. may be performed by utilizing a well-plate format.This format preferably allows the high-throughput performance of themethod for identifying polypeptides having cellobiohydrolase activity.

In a preferred embodiment, this method for identifying polypeptideshaving cellobiohydrolase activity is restricted to a method, wherein thepolypeptide(s) having cellobiohydrolase activity is one or morepolypeptide(s) as provided by this invention, such as having the IT50value given above, and/or being one of the specific variants of SEQ IDNO2 or SEQ ID NO: 5 as below.

Preferably, the steps e. to g. of the method for identifyingpolypeptides having cellobiohydrolase activity are performed as follows:

-   -   e. Incubating the expressed mutant protein with cellulosic        material;    -   f. Determining the amount of released sugar;    -   g. Selecting a mutant protein according to the amount of        released sugar.

In another embodiment, the method for identifying polypeptides havingcellobiohydrolase activity comprises the additional steps of:

-   -   h. Sequencing the selected mutant gene or protein;    -   i. Identifying the amino acid modification(s) by comparing the        sequence of the selected mutant protein with the amino acid        sequence of SEQ ID NO: 2.

In a particular embodiment, the method is further characterized bymeasuring the IT50 value of the obtained polypeptide. The IT50 value maybe measured as described in the examples below. Optionally, this may befollowed by a step of selection of those polypeptides, which display thea desired IT 50 value, such as at least 60° C., at least 62° C. and thelike. Thus, in this particular embodiment, the method is suitable foridentifying polypeptides exhibiting cellobiohydrolase activity and anelevated IT50 value, i.e. thermostable polypeptides withcellobiohydrolase activity.

The present invention further provides a method of preparing apolypeptide having cellobiohydrolase activity, comprising the steps:

-   -   a. Providing a polypeptide having cellobiohydrolase activity        comprising an amino sequence having at least 54% sequence        identity to the catalytic domain of SEQ ID NO: 2 (SEQ ID NO: 5)        (such as preferably, at least 60%, at least 62%, at least 64%,        at least 66%, at least 68% or at least 70%, whereby at least 68%        or at least 70% are the most preferred embodiments);    -   b. Identifying the amino acids of this polypeptide which        correspond to the amino acids which are modified with respect to        the amino acid sequence of SEQ ID NO: 2. as identified in        step i. of the method for identifying polypeptides having        cellobiohydrolase activity; and    -   c. Preparing a mutant polypeptide of the polypeptide provided in        step a. by carrying out the amino acid modification(s)        identified in step b. through site-directed mutagenesis.

In one embodiment, preferably, the polypeptide provided in step a. ofthe method of preparing a polypeptide having cellobiohydrolase activityis a wild type cellobiohydrolase derived from Trichoderma reesei.

The present invention further provides polypeptides havingcellobiohydrolase activity, which are obtainable by the method ofpreparing a polypeptide having cellobiohydrolase activity according tothe present invention.

Furthermore, the present invention provides a composition comprising apolypeptide and/or variants thereof of the present invention and one ormore cellulases, e.g. one or more endoglucanases and/or one or morebeta-glucosidases and/or one or more further cellobiohydrolases and/orone or more xylanases. “Cellulases” or “Cellulolytic enzymes” aredefined as enzymes capable of hydrolysing cellulosic substrates orderivatives or mixed feedstocks comprising cellulosic polymers. Suchenzymes are referred to as having “cellulolytic activity”, thus beingable to hydrolyze cellulose molecules from such material into smalleroligo- or monosaccharides. Cellulolytic enzymes include cellulases andhemicellulases, in particular they include cellobiohydrolases (CBHs),endoglucanases (EGs) and beta-glucosidases (BGLs).

The present invention further provides a polypeptide havingcellobiohydrolase activity, wherein the polypeptide comprises an aminoacid sequence having at least 80%, preferably at least 95%, morepreferably at least 98%, even more. preferably at least 99%, and mostpreferably 99, 6% sequence identity to SEQ ID NO: 5. Particularly, it ispreferred that such a polypeptide is a polypeptide wherein one or moreof the following amino acid residues of the sequence defined by SEQ IDNO: 5 are modified by substitution or deletion of: Q1. Q2, G4. A6. T7.A8. N10. P12. T15. A21. G23. S24. T26. T27. Q28. N29. G30. A31. V32.N37, W40. V41. G46. Y47. T48. N49. C50. T52. N54. D57. T59. Y60. D64.E65. A68. Q69. A72, V84. S86. S89. S90. K92. S99. Q109. D110. D111.I116. F117. K118. L119. L120. D129, V130. G139. A145. M146. V152. K154.Y155. N157. N158. K159. K163. G167. Q172. F179, I180. D181. E183. E187.G188. Q190. S192. S193; N194. I200. D202. H203. D211. V212, A221. P224.D228. T229. G231. T233. M234. S236. T243. Y244. S245. N246. D247. G251,F260. G266. K275. I276. I277. T280. L290. D293. G294. T295. T297. T299.S301. K304, F306. N310. S311. V313. I314. N318. D320. I321. T325. N327.T335. A340. F341. D343, T344. D345. D346. Q349. H350. A354. K355. A358.Q361. Q362. G363. M364. V367. D373, Y374. A375. A376. P386. T387. D390.T392. T393. P394. T400. P402. T403. D404. D410, N417. S418. T421. Y422and/or one or more insertions after positions G151. K159

In a preferred embodiment, the polypeptide having cellobiohydrolaseactivity with an amino acid sequence having at least 80% sequenceidentity to SEQ ID NO: 5 comprises one or more modified amino acidresidues of the sequence defined by SEQ ID NO: 5: Thus, the polypeptidegiven in SEQ ID NO: 5 may, by means of example, be modified as follows:Q1L, G4. A6G/V, T15S, Q28Q/R, W40R, D64N, E65K/V, A72V, S86T, K92K/R,V130I/V, V152A/E, Y155C, K159E, D181N, E183V/K,N194C/R/Y/D/K/I/L/G/Q/S/V, D202Y/N/G, P224L,T243I/R/Y/A/F/Q/P/D/V/W/L/M, Y244F/H, I277V, K304R, N310D, S311G/N,N318Y, D320V/E/N, T335I, T344M, D346G/A/E/V, Q349R/K, A358E,Y374C/P/R/H/S/A, A375D/N/Y/R/Q/L/V/E/G/T/M, T392C/D/K, T393A, D410G,Y422F.

More preferably, the polypeptide having cellobiohydrolase activitycomprises one or more modified amino acid residues of the sequencedefined by SEQ ID NO: 5 as indicated in the following Table 3. As saidabove, for the specific modifications of SEQ ID NO: 2. two or more ofsuch specific modifications may be combined with each other, such aspreferably two or more of the more preferred or most preferredmodifications may be combined with each other, and, which isparticularly preferred, two or more of the most preferred modificationsaccording to Table 3 may be combined with each other.

TABLE 3 Mutations with respect to SEQ ID NO: 5 Most Position PreferredMore Preferred Preferred Q1 L L L Q2 P, S P, S S G4 C C C A6 G, L, V G,L, V L T7 Q Q Q A8 S S S N10 T, D T, D T, D P12 Q T15 S A21 S, T, C S,T, C G23 A, D, N S24 T, C, N T, C, N T26 I, N I, N T27 S, Q S, Q Q28 L,K, R, N L, K, R, N K, R N29 T, Y T, Y G30 A A A31 S V32 G N37 S W40 R RV41 T G46 S Y47 S, F S, F T48 A N49 S C50 S T52 D N54 S D57 S T59 M Y60H D64 N N E65 V, M, K V, M, K V, M, K A68 T T Q69 K, R K, R A72 V, C V,C C V84 A S86 T T T S89 N S90 T, F K92 R R S99 T Q109 R D110 G, S, ND111 H, E I116 V, K, E F117 Y K118 A, T, Q A, T, Q L119 L, I L120 P, MD129 N V130 I G139 S A145 T M146 C G151 GCGRSG GCGRSG GCGRSG V152 A, EK154 R Y155 S, C, H S, C, H N157 S N158 D K159 E, KCGRNK KCGRNK KCGRNKK163 C G167 C Q172 Q F179 I I180 N D181 N N N E183 V, M, K V, M, K V, M,K E187 K G188 C Q190 L, K L, K S192 L, I, P, T, M L, I, P, T, M S193 L,P, T N194 G, L, I, V, S, C, G, L, I, V, S, C, K, R, D, Q, Y K, R, D, Q,Y I200 N, F D202 G, I, V, N, F, Y G, I, V, N, F, Y G, I, V, N, F, Y H203R R D211 G V212 L A221 V P224 L L L D228 N T229 A, S, M A, S, M G231 D DT233 S M234 L, I, V, T, K L, I, V, T, K S236 F, Y F, Y G, A, L, I, V, P,S, C, G, A, L, I, V, P, S, M, R, D, Q, F, Y, C, M, R, D, Q, F, Y, T243 WW Y244 H, F S245 T N246 S, K, D D247 N N G251 R F260 C G266 S K275 EI276 V I277 V T280 A L290 H D293 R, H G294 A T295 S T297 N T299 I, SS301 C K304 R F306 L, Y N310 D, E S311 G, D, N G, D, N G, D, N V313 II314 F N318 I, H, D, Y I, H, D, Y I, D, Y D320 I, V, E, N I, V, E, N I,V, N I321 N T325 A, I N327 Y T335 I I I A340 G, S, T G, S, T F341 C D343A T344 M M D345 E D346 G, A, V, E G, A, V, E G, A, V, E Q349 K, R K, RK, R H350 Y A354 T K355 Q Q A358 E Q361 R Q362 G, R, H G363 P M364 L, SV367 A D373 E Y374 A, P, S, C, R, H, D A, P, S, C, R, H, D A375 G, L, V,T, C, M, G, L, V, T, C, M, R, D, E, N, Q, Y R, D, E, N, Q, Y A376 T P386L, S T387 A, S A, S D390 G, E G, E T392 S, M, K S, M, K M T393 A, I, V,S A, I, V, S A, I, V P394 C T400 S P402 S T403 K D404 N D410 G N417 YS418 P T421 I Y422 F F F

Particularly preferred is a polypeptide as defined above, furthercharacterized by comprising a modification of SEQ ID NO: 5. which is aspecific modification as given in the following Table 3a. Each of thesepolypeptides defines a mutant version of the polypeptide given in SEQ IDNO: 5.

TABLE 3a specific mutants with respect to SEQ ID NO: 5: Mutant NumberMutants with respect to Seq. ID NO: 5 1 G4C, A72C, Q349K 2 G4C, A72C,T344M, Q349K 3 G4C, A72C, T344M, D346G, Q349R 4 G4C, A72C, D320V, Q349K5 G4C, A72C, P224L, F306Y, Q349R 6 G4C, A72C 7 A72V, D346A, T393A 8 G4C,A72C, Q349R 9 G4C, W40R, A72C, T344M, Q349K 10 A72V, D320V, D346A 11G4C, A72C, N194Y, T243L, Q349R, Y374S, A375R 12 G4C, E65V, A72C, Y244H,Q349R 13 G4C, A72C, D202G, D320N, Q349R, A358E 14 G4C, A72C, D320V,Q349R 15 G4C, A72C, Q349K, S86T 16 A72V, T335I, D346A, T393A 17 G4C,A72C, E183V, K304R, Q349K 18 G4C, A72C, T243G, Q349R, Y374P, A375M 19G4C, A72C, N194V, T243M, Q349R, Y374A, A375T 20 G4C, D64N, A72C, Q349R,A358E 21 G4C, A72C, Q349K, Q28R, S193T 22 G4C, A72C, E183K, Q349K 23G4C, A72C, S311N, Q349K 24 G4C, A72C, N194K, Q349R, Y374P, A375Q 25 G4C,A72C, D181N, Q349K 26 W40R, D320V, Q349K, T393A 27 W40R, T335I, D346A,T393A 28 Q1L, G4C, A72C, D181N, E183K, N327Y, Q349R 29 A72C, T335I,Q349R 30 G4C, A72C, N194K, T243P, Q349R, Y374H, A375E 31 G4C, A72V,Q349R, P462del 32 G4C, A72C, S236Y, Q349R 33 G4C, A72C, S311G, Q349K 34A72V, D320V, T335I, D346A, T393A 35 G4C, A72C, S86T, M234V, Q349K 36Q1L, G4C, Q28R, E65V, A72C, K159KCGRNK, D181N, E183M, P224L, S311G,D320I, D346E, Q349K, T393V, Y422F 37 G4C, A72C, G251R, Q349R 38 G4C,A72C, Q349K, D320V 39 G4C, A72C, E183K, Q349R 40 Q1L, G4C, A72C, H203R,Q349K 41 G4C, W40R, A72C, Q349K 42 G4C, A72C, Q349R, V367A 43 Q1L, G4C,A6V, C50S, A72C, I180N, D181N, E183K, Q349R 44 G4C, A72C, S311G, D320V,Q349K 45 Q1L, G4C, A72C, D181N, E183K, T243S, Q349R, P386S 46 Q1L, G4C,A72C, K154R, Q349K, T393I 47 G4C, A72C, N194G, T243F, Q349R, Y374P,A375R 48 A72V, D320V, D346A, T393A 49 A72C, Q349K 50 G4C, A72C, E183V,Q349K 51 G4C, A72C, E183K, N318Y, Q349K 52 W40R, A221V 53 G4C, A72C,K92R, Q349K 54 Q1L, G4C, A72C, S90T, D181N, E183K, Q349R 55 G4C, A72C,Q349R, Y422F 56 G4C, T48A, A72C, Q349R 57 E187K, D320V 58 G4C, S24N,E65K, A72C, Q349R 59 Q1L, G4C, A72C, S193P, Q349K 60 Q1L, G4C, A72C,delV152-K159, D181N, E183K, Q349R 61 Q1L, G4C, A72C, Q349K 62 Q1L, G4C,A72C, D181N, E183K, M234L, V313I, Q349R 63 Q1L, G4C, A72C, D181N, E183K,I200N, Q349R 64 G4C, A72C, N194K, T243Y, Q349R, A375N 65 Q1L, G4C, Q28R,A72C, Q349K 66 G4C, E65V, A72C, Q349R 67 D320V, Q349K 68 Q1L, G4C, A72C,S311G, Q349K 69 G4C, A72C, T243Q, Q349R, Y374P, A375M 70 Q1L, G4C, A72C,D320V, Q349R 71 Q1L, G4C, T15S, A72C, Y244F, Q349K 72 G4C, A72C, E183K,D346E, Q349R 73 Q1L, G4C, A72C, Q349K, T392M 74 G4C, A72C, D202N, S311N,Q349R 75 G4C, A72C, N194D, T243A, Q349R, Y374P, A375Y 76 G4C, A72C,N194Y, T243V, Q349R, Y374P 77 Q1L, G4C, A72C, Q349R 78 G4C, Q28R, E65K,A72C, S86T, D202N, H203R, S311N, D320I, A340G, D346A, Q349K, T393A,Y422F 79 G4C, A72C, D202N, Q349R 80 G4C, A72C, P224L, Q349R 81 Q1L, G4C,A72C, D181N, E183K, T229M, A340T, Q349R 82 Q1L, G4C, A72C, D181N, Q349R83 G4C, A72C, D320V, D346V, Q349K 84 Q1L, G4C, A72C, V152A, Q349K 85Q1L, G4C, A72C, Q349K, Y422F 86 G4C, A72C, D202V, D320V, Q349K 87 Q1L,G4C, A72C, Y155S, D181N, E183K, Q349R 88 Q1L, G4C, A72C, D181N, D247N,Q349K 89 Q1L, G4C, A68T, A72C, Q349K 90 Q1L, G4C, D64N, A72C, Q349K 91G4C, A72C, D181N, P224L, Q349K 92 G4C, A72C, N194I, T243Y, Q349R, Y374P,A375R 93 Q1L, G4C, A72C, E183K, Q349R 94 G4C, A72C, S311G, Q349R 95 Q1L,G4C, A72C, S311N, Q349K 96 Q1L, G4C, A72C, S86T, Q349R 97 Q1L, G4C,A72C, D181N, E183K, G231D, Q349R 98 Q1L, G4C, A72C, S89N, D181N, E183K,Q349R 99 Q1L, G4C, E65K, A72C, Q349K 100 Q1L, Q2P, G4C, W40R, E65M,A72C, S86T, S192L, D202N, H203R, S311D, D320I, T335I, D346G, Q349K,T392M, Y422F 101 Q1L, G4C, S24T, T26I, Q28R, N29Y, G30A, E65V, A68T,A72C, Y155C, D181N, E183M, Q190K, P224L, S311G, D320I, D346E, Q349K,T393V, Y422F 102 G4C, A72C, E183K, D202Y, N310D, Q349R 103 G4C, A72C,N194I, T243D, Q349R, Y374P, A375Y 104 Q1L, G4C, A72C, D181N, E183K,Q349R 105 G4C, Q28R, A72C, S86T, E183K, P224L, S311N, N318Y, T335I,D346G, Q349R, T393I 106 Q1L, G4C, A72C, D181N, Q349K 107 Q1L, G4C, A72C,D181N, E183K, T243I, N246D, Q349R 108 Q1L, G4C, G23N, A72C, D110G,I116V, L119I, D181N, E183K, D211G, D293R, N310D, Q349R, Q362R, G363P,M364S 109 Q1L, G4C, Q28R, E65V, A68T, A72C, Y155C, D181N, E183M, P224L,S311G, D320I, D346E, Q349K, T393V, Y422F 110 Q1L, G4C, A72C, S86T,D181N, E183K, Q349R, T393S 111 Q1L, G4C, G23N, A72C, V84A, D110G, D111H,I116E, F117Y, K118A, D181N, E183K, D293R, T295S, Q349R, M364L 112 Q1L,G4C, A72C, A145T, H203R, Q349K, T403K 113 Q1L, G4C, A72C, D181N, E183K,M234I, Q349R 114 Q1L, G4C, A72C, D181N, E183K, T297N, Q349R 115 G4C,Q28R, E65M, A72C, S86T, E183K, S192I, H203R, S311N, D346E, Q349K, T392M,T393A, Y422F 116 Q1L, G4C, A72C, D202N, Q349K 117 Q1L, G4C, A72C, S99T,D181N, E183K, Q349R 118 Q1L, G4C, A72C, I200F, Q349K 119 Q1L, G4C, A31S,A72C, D181N, E183K, Q349R 120 Q1L, G4C, Q28L, A72C, D181N, E183K, Q349R121 Q1L, G4C, A72C, D181N, E183K, T233S, Q349R 122 Q1L, G4C, A21T, A72C,D181N, E183K, Q349R 123 Q1L, G4C, A72C, D346V, Q349K 124 Q1L, G4C, Y47F,A72C, D181N, E183K, Q349R 125 Q1L, G4C, A72C, D181N, E183K, M234T, Q349R126 Q1L, G4C, A72C, N157S, D181N, E183K, Q349R 127 G4C, A72C, N194Q,T243V, Q349R, Y374P, A375Y 128 Q1L, G4C, A72C, D181N, E183K, I314F,Q349R 129 Q1L, G4C, A72C, Q349K, T392K 130 Q1L, G4C, A72C, D181N, E183K,M234V, Q349R 131 Q1L, G4C, A21S, A72C, D181N, E183K, Q349R 132 Q1L, G4C,Q28R, E65V, A72C, D181N, E183V, D228N, S311N, N318Y, D346E, Q349R, Y422F133 G4C, A72C, N194C, Q349R, Y374C 134 Q1L, G4C, A72C, D181N, E183K,Q349R, T400S 135 Q1L, G4C, T26I, A72C, D181N, E183K, Q349R 136 Q1L, G4C,A72C, D181N, E183K, N310D, Q349R, T392S 137 Q1L, G4C, A72C, D129N,D181N, E183K, Q190L, G266S, I276V, Q349R, P386L 138 Q1L, G4C, A72C,D181N, E183K, D202N, Q349R 139 Q1L, G4C, A72C, Y155C, D181N, E183K,Q349R 140 Q1L, G4C, Q28R, E65V, A72C, D181N, E183M, P224L, S311G, D320I,D346E, Q349K, T393V, Y422F 141 Q1L, G4C, A72C, D181N, E183K, N246K,Q349R 142 G4C, W40R, E65V, A72C, S86T, D181N, E183K, D202I, H203R,S311D, D320N, D346V, Q349R, T392M, T393A, Y422F 143 Q1L, G4C, A72C,Y155C, Q349K 144 Q1L, G4C, A68T, A72C, D181N, E183K, Q349R 145 Q1L, G4C,S24T, T26I, Q28R, N29Y, G30A, Y47F, E65V, A68T, A72C, Y155C, D181N,E183M, Q190K, P224L, T229M, G231D, M234T, S311G, D320I, A340S, D346E,Q349K, D390E, T393V, Y422F 146 Q1L, G4C, W40R, E65M, A72C, S86T, S192L,D202N, H203R, S311D, D320I, T335I, D346G, Q349K, T392M, Y422F 147 Q1L,G4C, A72C, S86T, D181N, E183K, D320V, Q349R 148 G4C, Q28K, A72C, S86T,E183M, D202N, P224L, S311G, N318Y, D320N, D346A, Q349R, T392M, T393I 149Q1L, Q2P, G4C, Q28R, W40R, E65K, A72C, D181N, S192L, D202I, H203R,P224L, S311G, D320I, D343A, D346A, Q349K 150 Q1L, G4C, Q28R, E65K, A72C,E183M, D202I, P224L, D320N, D346V, Q349K, T392M, T393V, Y422F 151 Q1L,G4C, Q28R, E65V, A72C, S86T, E183K, D202V, S311G, N318Y, D320I, D346G,Q349K, T393V, Y422F 152 Q1L, G4C, W40R, E65M, A72C, D181N, E183K, S192P,D202N, P224L, S311D, N318Y, D320V, D346G, Q349K, T392M 153 Q1L, G4C,E65V, A72C, D181N, E183K, P224L, S311G, D320N, D346G, Q349R, T392M,T393I 154 Q1L, G4C, Q28R, E65V, A72C, G151GCGRSG, D181N, E183M, P224L,S311G, D320I, D346E, Q349K, T393V, Y422F 155 Q1L, G4C, E65M, A72C,D181N, E183M, D202Y, P224L, S311D, N318Y, D320I, T335I, D346A, Q349K,T392M, T393I 156 Q1L, G4C, G23A, A72C, D110S, D111H, I116V, F117Y,K118A, L120M, D181N, E183K, D293H, G294A, N310E, Q349R, Q362G, M364S 157Q1L, G4C, A72C, D181N, E183K, Q349R, T421I 158 G4C, Q28K, E65M, A72C,S86T, V152A, D181N, E183V, S192L, D202N, S311N, D320N, D346E, Q349R,T387A, T392M, T393I, Y422F 159 Q1L, G4C, W40R, E65V, A72C, S86T, E183V,G188C, S192T, D202Y, H203R, P224L, S311N, D320V, D346E, Q349K, T393V,Y422F 160 Q1L, G4C, E65M, A72C, S86T, E183M, D202N, P224L, T335I, D346G,Q349K, T392M, T393A 161 Q1L, G4C, Q28R, E65V, A72C, D181N, E183M, P224L,S311G, D320I, A340S, D346E, Q349K, D390E, T393V, Y422F 162 G4C, W40R,A72C 163 Q1L, G4C, W40R, E65K, A72C, S86T, E183K, S192L, D202Y, P224L,D320I, D346E, Q349R 164 Q1L, G4C, A21T, T26I, Q28R, E65V, A68T, A72C,Y155C, D181N, E183M, Q190K, D202N, P224L, S311G, N318Y, D320I, D346E,Q349K, T393V, Y422F 165 Q1L, G4C, Q28R, E65V, A72C, S86T, D181N, D202N,P224L, S311G, N318Y, D320I, T335I, D346E, Q349K, T393V, Y422F 166 Q1L,G4C, Q28R, E65V, A72C, E183M, S311G, D346E, Q349K, T393V, Y422F 167 Q1L,G4C, T7Q, A8S, N10T, S24T, T27Q, Q28R, N29T, V41T, G46S, Y47S, T52D,D57S, D64N, E65V, Q69K, A72C, D181N, P224L, S311G, N318Y, D320N, D346A,Q349K, T392M, T393I, Y422F 168 Q1L, G4C, Q28R, E65V, A72C, K159KCGRNK,E183M, P224L, S311G, D320I, D346E, Q349K, T393V, Y422F 169 Q1L, G4C,Q28K, W40R, E65V, A72C, D181N, E183V, S192P, D202V, H203R, S311G, D320N,D346E, Q349K, T392M, T393A, Y422F 170 Q1L, G4C, Q28R, E65V, A72C, G139S,D181N, E183M, P224L, S311G, D320I, D346E, Q349K, T393V, Y422F 171 Q1L,G4C, Q28R, E65V, A72C, S86T, D181N, E183M, D202N, P224L, S311G, N318Y,T335I, D346E, Q349K, T393V, Y422F 172 Q1L, G4C, Q28R, E65K, A72C, D181N,S311N, N318Y, D346E, Q349K, T393V, Y422F 173 Q1L, G4C, W40R, A72C,S192L, D202N, H203R, P224L, S311N, D320I, T335I, D346V, Q349R, T393I 174Q1L, G4C, Q28K, E65K, A72C, E183M, D202N, P224L, T229S, S311G, D320I,T335I, D346V, Q349R, T393V 175 Q1L, G4C, Q28R, G30A, E65M, A72C, D181N,D202N, P224L, S311D, N318Y, D346E, Q349K, T392M, T393V, Y422F 176 Q1L,G4C, Q28R, E65V, A72C, S68T, E183M, S311N, T335I, D346E, Q349K, T393V,Y422F 177 Q1L, G4C, Q28K, E65K, A72C, D181N, D202N, S311N, N318Y, T335I,D346E, Q349K, T393V, Y422F 178 T243C, A375C, N194C, Y374C 179 Q1L, G4C,Q28K, E65K, A72C, S86T, E183M, S311G, D346V, Q349R, T392M, T393V, Y422F180 Q1L, G4C, Q28R, E65V, A72C, D181N, E183M, S311G, D346E, Q349K,T393V, Y422F 181 Q1L, G4C, Q28R, E65V, A72C, S86T, D181N, D202N, S311N,N318Y, D320I, T335I, D346E, Q349K, T393I, Y422F 182 Q1L, G4C, Q28R,E65K, A72C, S86T, E183K, D202N, P224L, S311G, T335I, D346A, Q349K,T393V, Y422F 183 Q1L, G4C, Q28R, E65V, A72C, L120P, D181N, E183M, P224L,S311G, D320I, D346E, Q349K, T393V, Y422F 184 Q1L, G4C, Q28K, E65V, A72C,S86T, E183M, S311N, N318H, D320V, D346V, Q349K, T392M, T393A 185 Q1L,G4C, Q28K, E65V, A72C, D202N, H203R, S311G, T335I, D346V, Q349K, T393A186 Q1L, G4C, Q28R, E65V, A72C, E183M, S311G, D320I, D346E, Q349K,T393V, Y422F 187 Q1L, G4C, Q28R, E65K, A72C, D181N, D202N, H203R, P224L,S311D, D346G, Q349K 188 Q1L, G4C, Q28R, E65M, A72C, D181N, E183M, S311G,N318Y, D320I, T335I, D346E, Q349K, D390E, T393V, Y422F 189 Q1L, G4C,T27S, Q28R, E65V, Q69R, A72C, L120P, D181N, E183M, D202N, D247N, S311G,D346E, Q349K, K355Q, T387S, T393V, Y422F 190 Q1L, G4C, Q28R, E65K, A72C,E183V, S192T, D202N, S311G, D320V, D346A, Q349K 191 Q1L, G4C, W40R,E65V, A72C, S86T, E183M, D202N, P224L, S311G, D320V, D346E, Q349R, T393I192 Q1L, G4C, Q28N, E65K, A72C, D181N, E183M, D202N, H203R, S311G,N318Y, D320N, Q349R, T393V, Y422F 193 Q1L, G4C, Q28R, V41T, G46S, Y47S,T52D, E65V, A72C, D181N, P224L, S311D, N318Y, D320N, D346A, Q349K,T392M, T393I, Y422F 194 Q1L, G4C, Q28R, E65K, A72C, D181N, E183M, P224L,S311G, D320I, D346E, Q349K, T393V, Y422F 195 Q1L, G4C, Q28R, W40R, A72C,S86T, D181N, S192T, D202N, P224L, S311G, N318Y, D320V, D346E, Q349K,T392M, T393I 196 Q1L, G4C, A21T, T26I, Q28R, G30A, E65V, A68T, A72C,Y155C, D181N, Q190K, D202N, P224L, T229M, S311G, N318Y, D320I, T335I,D346E, Q349K, T393V, Y422F 197 Q1L, G4C, Q28K, W40R, A72C, S86T, D181N,E183M, S192I, D202Y, T299S, S311N, N318Y, D320I, D346V, Q349R, T393I 198Q1L, G4C, Q28R, E65V, A72C, D181N, E183M, P224L, T229M, G231D, M234T,S311G, D320I, D346E, Q349K, T393V, Y422F 199 Q1L, G4C, S24T, T26I, Q28R,N29Y, G30A, E65V, A68T, A72C, Y155C, D181N, E183M, Q190K, P224L, T229M,G231D, M234T, S311G, D320I, A340S, D346E, Q349K, D390E, T393V, Y422F 200Q1L, G4C, Q28R, E65K, A72C, E183M, P224L, S311G, D346E, Q349K, T393V,Y422F 201 Q1L, G4C, Q28R, E65V, A72C, S86T, D181N, E183M, D202N, P224L,S311G, N318Y, D346E, Q349K, T393V, Y422F 202 Q1L, G4C, Q28R, W40R, E65V,A72C, S86T, S192T, D202V, H203R, S311N, N318Y, D346A, Q349K, T392M,T393A, Y422F 203 Q1L, G4C, Q28K, E65K, A72C, D181N, H203R, P224L, S311G,D320V, D346G, Q349K, T392M, T393I 204 Q1L, G4C, Q28R, E65K, A72C, E183M,S311G, D346E, Q349K, T393V, Y422F 205 Q1L, G4C, Q28R, E65M, A72C, D181N,E183M, S311G, N318Y, D346E, Q349K, T393V, Y422F 206 Q1L, G4C, Q28R,E65V, A72C, E183K, D202I, P224L, S311G, D320I, Q349K, T393V 207 Q1L,G4C, Q28K, A72C, S86T, D181N, E183V, S192T, D202N, P224L, S311G, N318Y,D320V, D346G, Q349R, T392M, Y422F 208 G4C, Q28R, E65K, A72C, S86T,D181N, E183M, S192L, D202N, H203R, P224L, S311D, D346E, Q349R, T392M,T393A, Y422F 209 Q1L, G4C, W40R, E65K, A72C, Q109R, D181N, E183M, S192I,D202I, H203R, S245T, D346A, Q349R, T393A, Y422F 210 Q1L, G4C, Q28K,E65M, A72C, D181N, D202N, S311N, T335I, D346E, Q349K, T393I, Y422F 211Q1L, G4C, N10D, Q28R, E65V, Q69R, A72C, D181N, E183M, P224L, S311G,D320I, D346E, Q349R, T393V, N417Y, Y422F 212 Q1L, G4C, Q28R, W40R, E65V,A72C, D202Y, H203R, P224L, T299I, N318Y, D320V, D346A, Q349K, T392M,T393A 213 G4C, Q28R, E65V, A72C, E183M, D202N, P224L, S311G, N318Y,D320V, D346G, Q349K, T392M, Y422F 214 Q1L, G4C, Q28K, A72C, S86T, E183K,S311G, D320V, D346A, Q349R, T392M, T393I, Y422F 215 Q1L, G4C, Q28R,E65M, A72C, D181N, P224L, S311N, D320N, T335I, 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S311N, D320I, T335I, D346E, Q349K,T393V, Y422F 308 Q1L, G4C, Q28K, E65K, A72C, D181N, D202N, P224L, S311G,N318Y, T335I, D346E, Q349K, T393V, Y422F 309 Q1L, G4C, Q28R, E65V, A72C,D181N, E183M, D202N, P224L, S311G, D320I, D346E, Q349K, T393V, Y422F 310Q1L, G4C, Q28R, E65V, A72C, D181N, E183M, D202N, S311N, T335I, D346A,Q349K, T393V, Y422F 311 Q1L, G4C, Q28K, E65K, A72C, S86T, E183M, P224L,S311G, N318Y, D320I, T335I, D346E, Q349K, T393V, Y422F 312 Q1L, G4C,A21T, T26I, Q28R, G30A, E65V, A68T, A72C, Y155C, D181N, Q190K, D202N,T229M, S311G, D320I, T335I, D346E, Q349K, T393V, Y422F 313 Q1L, G4C,Q28K, E65M, A72C, D181N, E183M, S311G, T335I, D346E, Q349K, T393V, Y422F314 Q1L, G4C, Q28R, E65V, A72C, D181N, E183M, P224L, S311G, D320I,D346E, Q349K, T393V, D404N, Y422F 315 Q1L, G4C, Q28R, E65V, A72C, E183M,D202N, P224L, S311G, N318Y, T335I, D346E, Q349K, T393V, Y422F 316 Q1L,G4C, Q28R, E65V, A72C, D181N, E183M, P224L, T280A, S311G, D320I, D346E,Q349K, T393V, Y422F 317 Q1L, G4C, Q28R, E65V, Q69R, A72C, D181N, E183M,P224L, S311G, D320I, D346E, Q349K, T393V, Y422F 318 Q1L, G4C, Q28R,E65V, A72C, D181N, E183M, P224L, K275E, S311G, D320I, D346E, Q349K,T393V, Y422F 319 Q1L, G4C, Q28K, E65V, A72C, E183K, D202N, P224L, S311N,D320I, D346E, Q349K, T393V, Y422F 320 Q1L, G4C, Q28K, E65V, A72C, D181N,E183M, S311G, N318Y, D320I, D346A, Q349K, T393I, Y422F 321 Q1L, G4C,S24T, T26I, Q28R, N29Y, G30A, E65V, A72C, D181N, E183M, P224L, S311G,D320I, D346E, Q349K, T393V, Y422F 322 Q1L, G4C, Q28R, E65M, A72C, E183M,S311G, D320I, D346A, Q349K, T393V, Y422F 323 Q1L, G4C, Q28R, E65V, A72C,D181N, E183M, D202N, S311G, D346E, Q349K, T393I, Y422F 324 Q1L, G4C,Q28R, E65V, A72C, E183M, P224L, S311G, D320I, D346E, Q349K, T393V, Y422F325 Q1L, G4C, Q28K, E65K, A72C, D181N, E183M, S311G, N318Y, D320I,T335I, D346E, Q349K, T393V, Y422F 326 Q1L, G4C, Q28R, E65V, A72C, S311G,N318Y, D320I, T335I, D346E, Q349K, T393V, Y422F 327 Q1L, G4C, S24C,Q28R, G30A, E65V, A72C, Y155C, D181N, Q190K, D202N, P224L, S311G, T335I,D346E, Q349K, D390E, T393V, Y422F 328 Q1L, G4C, Q28R, E65V, A72C, D181N,E183M, P224L, S311G, D320I, D346E, Q349K, T393V, P402S, Y422F 329 Q1L,G4C, Q28R, E65K, A72C, E183M, D202N, P224L, S311N, N318Y, T335I, D346E,Q349K, T393V, Y422F 330 Q1L, G4C, A21T, T26I, Q28R, N29Y, E65V, A72C,Y155C, D181N, D202N, P224L, M234T, S311G, D320I, D346E, Q349K, D390E,T393V, Y422F 331 Q1L, G4C, Q28R, E65K, A72C, S86T, D202N, P224L, S311D,D320N, T335I, D346V, Q349K, T392M, T393V, Y422F 332 Q1L, G4C, Q28R,E65V, A72C, S86T, E183K, D202N, S311G, N318Y, D320I, D346A, Q349K,T393V, Y422F 333 Q1L, G4C, W40R, E65M, A72C, S86T, E183V, S192L, D202I,H203R, P224L, S311G, N318Y, D320V, D346A, Q349K 334 Q1L, G4C, Q28R,E65V, A72C, D181N, E183M, P224L, S311G, D346E, Q349K, T393V, Y422F 335Q1L, G4C, Q28R, E65V, A72C, K118Q, D181N, E183M, P224L, D247N, S311G,D320I, D346E, Q349K, T393V, Y422F 336 Q1L, G4C, Q28R, E65V, A72C, D181N,E183M, D202N, P224L, S311N, D320I, D346E, Q349K, T393V, Y422F 337 Q1L,G4C, Q28R, E65V, A72C, S86T, D202N, P224L, S311G, N318Y, T335I, D346E,Q349K, T393V, Y422F 338 Q1L, G4C, Q28K, E65M, A72C, D181N, E183M, D202N,S311G, N318Y, D346A, Q349K, T393V, Y422F 339 Q1L, G4C, Q28R, E65V, A72C,D181N, E183M, P224L, S311G, D320I, D346E, Q349K, T393V, Y422F,P454PVRPQP 340 Q1L, G4C, Q28R, A72C, D181N, E183V, S192M, D202N, P224L,S311D, N318Y, D320N, D346E, Q349R, T393V, Y422F 341 Q1L, G4C, Q28R,A72C, S86T, D181N, S192L, D202N, P224L, S311G, N318Y, D320V, T335I,D345E, D346A, Q349K, Y422F 342 Q1L, G4C, Q28R, E65V, A72C, S86T, D181N,E183K, P224L, S311G, N318Y, D320I, D346A, Q349K, T393V, Y422F 343 Q1L,G4C, Q28R, G30A, E65V, A68T, A72C, D181N, E183M, D202N, P224L, D346E,Q349K, T393V, Y422F 344 Q1L, G4C, Q28R, E65V, A72C, K92R, L120P, D181N,E183M, D202N, S236F, T280A, S311G, D346E, Q349K, K355Q, T387S, T393V,P402S, D404N, Y422F 345 Q1L, G4C, Q28R, E65V, A72C, D181N, D202N, S236F,S311G, T335I, D346E, Q349K, T387S, T393V, Y422F 346 Q1L, G4C, Q28R,E65M, A72C, D181N, E183K, D202N, P224L, S311G, D320I, D346E, Q349K,T393V, Y422F 347 Q1L, G4C, A21T, T26I, Q28R, G30A, E65V, A68T, A72C,Y155C, D181N, E183M, Q190K, D202N, S311G, D320I, D346E, Q349K, T393V,Y422F 348 Q1L, G4C, Q28R, E65V, A72C, D181N, D202N, S311G, T335I, D346A,Q349K, T393I, Y422F 349 Q1L, G4C, Q28R, E65V, A72C, D181N, E183M, P224L,D247N, S311G, D320I, D346E, Q349K, T387S, T393V, Y422F 350 Q1L, G4C,Q28R, E65V, A72C, D181N, D202N, P224L, S311G, D346A, Q349K, T393V, Y422F351 Q1L, G4C, Q28R, E65V, A72C, K92R, K118Q, D181N, E183M, D202N, S236F,S311N, N318I, D346E, Q349K, K355Q, T387S, T393V, D404N, Y422F 352 Q1L,G4C, T27S, Q28R, E65V, Q69R, A72C, K118Q, D181N, E183M, D202N, D247N,I277V, S311G, T335I, D346E, Q349K, K355Q, T393V, Y422F 353 Q1L, G4C,Q28R, G30A, E65V, A72C, Y155C, D181N, E183M, D202N, M234T, S311G, D346E,Q349K, T393V, Y422F 354 Q1L, G4C, Q28R, E65V, Q69R, A72C, K92R, D181N,D202N, D247N, I277V, S311G, N318D, T335I, D346E, Q349K, K355Q, T387S,T393V, Y422F 355 Q1L, G4C, T27S, Q28R, E65V, Q69R, A72C, D181N, D202N,S236F, I277V, S311G, T335I, D346E, Q349K, K355Q, T387S, T393V, Y422F 356Q1L, G4C, N10D, Q28R, E65V, Q69R, A72C, K92R, K118Q, D181N, E183M,D202N, S311G, T335I, D346E, Q349K, T387S, T393V, Y422F 357 Q1L, G4C,A21T, Q28R, G30A, E65V, A72C, Y155C, D181N, E183M, D202N, M234T, S311G,T335I, D346E, Q349K, T393V, Y422F 358 Q1L, G4C, Q28R, E65V, A72C, D181N,E183M, P224L, S311G, D320I, D346E, Q349K, T393V, Y422F, T457TAAATT 359Q1L, G4C, T26I, Q28R, E65V, A68T, A72C, Y155C, D181N, Q190K, D202N,P224L, M234T, S311G, D320I, D346E, Q349K, D390E, T393V, Y422F 360 Q1L,G4C, Q28K, E65V, A72C, S86T, E183K, S192L, D202N, D320N, T335I, D346A,Q349R, T393A, Y422F 361 Q1L, G4C, Q28R, E65V, A72C, D181N, E183M, P224L,S236F, S311G, D320I, D346E, Q349K, T393V, Y422F 362 Q1L, G4C, T26I,Q28R, E65V, A68T, A72C, Y155C, D181N, D202N, M234T, S311G, T335I, D346E,Q349K, T393V, Y422F 363 Q1L, G4C, A21T, T26I, Q28R, N29Y, E65V, A72C,Y155C, D181N, E183M, D202N, P224L, M234T, S311G, D320I, T335I, A340S,D346E, Q349K, D390E, T393V, Y422F 364 Q1L, G4C, Q28R, E65K, A72C, D202I,P224L, S311G, N318Y, D320V, T335I, D346A, Q349R, T392M, T393A 365 Q1L,G4C, E65M, A72C, S86T, E183V, D202I, P224L, S311G, N318Y, D320V, D346A,Q349K 366 Q1L, G4C, N10D, Q28R, E65V, A72C, K92R, D181N, E183M, D202N,S311G, D346E, Q349K, T387S, T393V, Y422F 367 Q2S, G4C, A6L, T7Q, A8S,N10T, Q28K, E65M, A72C, S86T, E183M, D202N, P224L, S311G, D320I, T335I,D346E, Q349K, T393V, Y422F 368 Q2S, G4C, A6L, T7Q, A8S, N10T, S24T,T26I, Q28R, N29Y, G30A, E65V, A72C, E183M, P224L, S311G, D320I, D346E,Q349K, T393V, Y422F 369 Q1L, G4C, Q28R, E65V, A72C, K92R, D181N, E183M,P224L, I277V, S311G, D320I, D346E, Q349K, T393V, Y422F 370 Q2S, G4C,A6L, T7Q, A8S, N10T, Q28R, E65V, A72C, E183M, P224L, S311G, D320I,D346E, Q349K, T393V, Y422F 371 Q2S, G4C, A6L, T7Q, A8S, N10T, Q28R,E65V, A72C, G151GCGRSG, E183M, P224L, S311G, D320I, D346E, Q349K, T393V,Y422F 372 Q2S, G4C, A6L, T7Q, A8S, N10T, Q28R, E65V, A72C, K159KCGRNK,E183M, P224L, S311G, D320I, D346E, Q349K, T393V, Y422F 373 Q2S, G4C,A6L, T7Q, A8S, N10T, Q28K, E65K, A72C, E183M, S311G, N318Y, D320I,T335I, D346E, Q349K, T393V, Y422F 374 Q2S, G4C, A6L, T7Q, A8S, N10T,Q28K, E65V, A72C, E183M, D202N, P224L, S311G, T335I, D346E, Q349K,T393V, Y422F 375 Q2S, G4C, A6L, T7Q, A8S, N10T, Q28R, E65M, A72C, S86T,E183M, S311N, N318Y, T335I, D346E, Q349K, T393V, Y422F 376 Q2S, G4C,A6L, T7Q, A8S, N10T, Q28R, E65M, A72C, E183M, S311G, N318Y, D346E,Q349K, T393V, Y422F 377 Q2S, G4C, A6L, T7Q, A8S, N10T, Q28R, E65V, A72C,E183M, D202N, P224L, S311G, N318Y, T335I, D346E, Q349K, T393V, Y422F 378Q2S, G4C, A6L, T7Q, A8S, N10T, Q28R, E65V, A72C, E183M, P224L, S311G,N318Y, T335I, D346A, Q349K, T393V, Y422F 379 Q2S, G4C, A6L, T7Q, A8S,N10T, Q28R, E65V, A72C, P224L, S311D, N318Y, D320N, D346A, Q349K, T392M,T393I, Y422F 380 Q2S, G4C, A6L, T7Q, A8S, N10T, Q28R, E65K, A72C, S311N,N318Y, D346E, Q349K, T393V, Y422F 381 Q2S, G4C, A6L, T7Q, A8S, N10T,Q28K, N29Y, E65K, A72C, S311G, T335I, D346E, Q349K, T393V, Y422F 382Q2S, G4C, A6L, T7Q, A8S, N10T, S24T, T26I, Q28R, N29Y, G30A, E65V, A68T,A72C, E183M, Q190K, P224L, S311G, D320I, D346E, Q349K, T393V, Y422F 383Q2S, G4C, A6L, T7Q, A8S, N10T, S24T, T26I, Q28R, N29Y, G30A, Y47F, E65V,A68T, A72C, E183M, Q190K, P224L, T229M, G231D, M234T, S311G, D320I,A340S, D346E, Q349K, D390E, T393V, Y422F 384 Q1L, G4C, A72C, S86T,D181N, E183K, D202V, P224L, S311G, D320V, D346E, Q349R, T393A, Y422F 385Q1L, G4C, Q28R, E65V, A72C, K159CGRNKE183M, P224L, S311G, D320I, D346E,Q349K, T393V, Y422F 386 Q2S, G4C, A6L, T7Q, A8S, N10T, S24T, Q28R, E65K,A72C, D202N, S311G, T335I, D346E, Q349K, T393I, Y422F 387 Q2S, G4C, A6L,T7Q, A8S, N10T, Q28R, E65M, A72C, E183K, D202N, P224L, S311G, D320I,D346E, Q349K, T393V, Y422F 388 Q1L, G4C, Q28K, E65M, A72C, S86T, E183M,D202N, P224L, S311G, D320I, T335I, D346E, Q349K, T393V, Y422F 389 Q1L,G4C, S24T, T26I, Q28R, N29Y, G30A, E65V, A72C, E183M, P224L, S311G,D320I, D346E, Q349K, T393V, Y422F 390 Q1L, G4C, Q28R, E65V, A72C, D181N,E183M, P224L, S311G, D320I, D346E, Q349K, T393V, Y422F, Q1L, G4C, Q28R,E65V, A72C, E183M, P224L, S311G, D320I, D346E, Q349K, T393V, Y422F 391Q1L, G4C, Q28R, E65V, A72C, G151GCGRSG, E183M, P224L, S311G, D320I,D346E, Q349K, T393V, Y422F 392 Q1L, G4C, Q28R, E65K, A72C, S311N, N318Y,D346E, Q349K, T393V, Y422F 393 Q1L, G4C, S24T, T26I, Q28R, N29Y, G30A,E65V, A68T, A72C, E183M, Q190K, P224L, S311G, D320I, D346E, Q349K,T393V, Y422F 394 Q1L, G4C, S24T, T26I, Q28R, N29Y, G30A, Y47F, E65V,A68T, A72C, E183M, Q190K, P224L, T229M, G231D, M234T, S311G, D320I,A340S, D346E, Q349K, D390E, T393V, Y422F 395 Q1L, G4C, A21T, T26I, Q28R,N29Y, G30A, E65V, A68T, A72C, D181N, Q190K, D202N, T229M, M234T, S311G,D320I, A340S, D346E, Q349K, D390E, T393V, Y422F 396 Q1L, G4C, N10D,T27S, Q28R, E65V, A72C, K92R, K118Q, D181N, D202N, S236F, I277V, S311G,D346E, Q349K, K355Q, T387S, T393V, D404N, N417Y, Y422F 397 Q1L, G4C,A21T, T26I, Q28R, G30A, E65V, A68T, A72C, Y155C, D181N, Q190K, D202N,P224L, T229M, S311N, D320I, D346E, Q349K, T393V, Y422F 398 Q1L, G4C,N10D, T27S, Q28R, E65V, A72C, K92R, K118Q, D181N, E183M, D202N, D247N,S311G, D320I, T335I, D346E, Q349K, T393V, Y422F 399 Q1L, G4C, T26I,Q28R, G30A, E65V, A72C, Y155C, D181N, D202N, T229M, G231D, S311G, T335I,D346E, Q349K, T393V, Y422F 400 Q1L, G4C, S24R, T26I, Q28R, G30A, E65V,A72C, D181N, E183M, D202N, P224L, S311N, T335I, D346E, Q349K, D390E,T393V, Y422F 401 Q1L, G4C, N10D, T27S, Q28R, E65V, Q69R, A72C, D181N,E183M, D202N, S236F, D247N, I277V, S311N, T335I, D346E, Q349K, T387S,T393V, N417Y, Y422F 402 Q1L, G4C, A21T, Q28R, E65V, A72C, Y155C, D181N,D202N, P224L, S311G, N318Y, D346E, Q349K, D390E, T393V, Y422F 403 Q1L,G4C, T26I, Q28R, N29Y, E65V, A68T, A72C, Y155C, D181N, E183M, D202N,P224L, M234T, S311N, N318Y, D346E, Q349K, T393V, Y422F 404 Q1L, G4C,A21T, T26I, Q28R, N29Y, E65V, A68T, A72C, Y155C, D181N, D202N, P224L,T229M, M234T, S311G, N318Y, D346E, Q349K, T393V, Y422F 405 Q1L, G4C,N10D, Q28R, E65V, A72C, K92R, K118Q, D181N, E183M, D202N, P224L, S236F,D247N, F306L, S311G, D346E, Q349K, K355Q, T393V, D404N, N417Y, Y422F 406Q1L, G4C, N10D, Q28R, E65V, A72C, K92R, K118Q, D181N, E183M, D202N,S236F, D247N, S311G, N318I, T335I, D346E, Q349K, K355Q, T393V, Y422F 407G4C, Q28K, E65M, A72C, S86T, G151GCGRSG, E183M, D202N, P224L, S311G,T335I, D346E, Q349K, T393V, Y422F 408 G4C, Q28K, E65M, A72C, S86T,K159KCGRNK, E183M, D202N, P224L, S311G, T335I, D346E, Q349K, T393V,Y422F 409 G4C, Q28K, E65M, A72C, S86T, G151GCGRSG, K159KCGRNK, E183M,D202N, P224L, S311G, T335I, D346E, Q349K, T393V, Y422F 410 Q1L, G4C,Q28K, E65V, A72C, D181N, E183M, D202N, P224L, S311G, T335I, D346E,Q349K, T393V, Y422F 411 Q1L, G4C, Q28K, E65V, A72C, S86T, D181N, E183K,S192L, D202V, S311G, D320I, D346V, Q349R, T393A, Y422F 412 Q1L, G4C,Q28R, E65V, A72C, S86T, D181N, D202N, P224L, S311N, N318Y, D320I, D346A,Q349K, T393V, Y422F 413 Q1L, G4C, A21T, T26I, Q28R, G30A, E65V, A68T,A72C, D181N, E183M, D202N, T229M, S311G, A340S, D346E, Q349K, T393V,Y422F

Furthermore, the present invention provides a polypeptide havingcellobiohydrolase activity comprising an amino acid sequence having atleast 85%, preferably at least 95%, more preferably at least 98%, evenmore preferably at least 99%, and most preferably 100% sequence identityto SEQ ID NO:12 wherein one or more of the following amino acid residuesof the sequence defined by SEQ ID NO: 12 are modified by substitution ordeletion: Q1. S2. P12. T15. S21. G23. T26. Q28. T29. G30. V32. N37. W40.T48. C50. N54. L60. E65, K69. V84. S90. D114. E119. F120. T121. L122.L123. D132. V133. G142. S148. M149, V155. Y158. N161. T162. K166. G170.Q175. F182. I183. G191. I203. D214. I215. A224, T231. G234. I237. S248.G254. W263. G269. L282. T285. G298. Y303. N307. G308. T310, E317. L318.S322. N324. G340. S341. D345. S357. M360. V363. D369. A372. P382. S388,T389. P390. T399. S400. Q406. N413. F423. P425. I426. G427. T429. P432.G435. N436, P437. G439. N441. R442. T444. T445. T446. T447. R449. P450.A451. T452. T453. S456, S457. P458. G463. P464. S467. H459. C468. G470.G472. S474. P476. V478. C479. S481, G482. T484. V487. L488. N489. Y491.Y492. Q494. C495. L496. Preferably, this polypeptide comprises an aminoacid sequence with at least 54%, preferably at least 56%, morepreferably at least 58%, particularly preferably at least 60%, such asat least 62%, particularly at least 64%, such as at least 66%, and mostpreferably preferably at least 68% sequence identity to SEQ ID NO: 5.This polypeptide also preferably lies within the embodiment as definedabove, wherein the polypeptide is temperature stable, i.e. has a highIT50 value, such as defined above, for example 62° C. or more, asdescribed above (for more embodiments, see above, in relation to thedefinition of variants of SEQ ID NO: 5). The skilled person willrecognize that SEQ ID NO 12: has about 68% identity with SEQ ID NO: 5.Therefore, a polypeptide derived from the polypeptide defned by SEQ IDNO: 12. which is differs from the polypeptide defined by SEQ ID NO: 12for example by exchange of one amino acid for another, such as Q1A, forexample, is a polypeptide which also has a significant degree ofidentity with SEQ ID: NO: 5. i.e. at least 66% or more, as definedabove. Thus, the skilled person can readily recognize the commoninventive concept of this invention, particular when taking intoconsideration the temperature stability of the polypeptides of thisinvention.

In a preferred embodiment, the polypeptide having cellobiohydrolaseactivity comprises an amino acid sequence having at least 85% sequenceidentity to SEQ ID NO: 12. wherein the polypeptide has the amino acidsequence of SEQ ID NO: 12 wherein one or more of the following aminoacid residues are modified by substitution or deletion: Q1. S2. P12.T15. S21, G23. T26. Q28. T29. G30. V32. N37. W40. T48. C50. N54. L60.E65. K69. V84. S90. D114, E119. F120. T121. L122. L123. D132. V133.G142. S148. M149. V155. Y158. N161. T162, K166. G170. Q175. F182. I183.G191. I203. D214. I215. A224. T231. G234. I237. S248, G254. W263. G269.L282. T285. G298. Y303. N307. G308. T310. E317. L318. S322. N324, G340.S341. D345. S357. M360. V363. D369. A372. P382. S388. T389. P390. T399.S400, Q406. N413. F423. P425. T426. G427. T429. P432. G435. N436. P437.G439. N441. R442, T444. T445. T446. T447. R449. P450. A451. T452. T453.S456. S457. P458. G463. P464, S467. H459. C468. G470. G472. S474. P476.V478. C479. S481. G482. T484. V487. L488, N489. Y491. Y492. Q494. C495.L496

Exchange with respect to SEQ ID NO: 12 1 Q1L 2 T15S 3 Q28R 4 W40R 5 C72V6 V133I 7 V155A, E 8 Y158C 9 T162E 10 Y247F, H 11 N307D 12 G308N 13E317V, N 14 S341M 15 D345R, K 16 Y370P, R, H, S, A 17 T389A 18 Q406G 19N441D 20 R442S, G 21 T452A 22 S456L, P 23 P458L, del 24 G459D 25 H464L,Q, R 26 V478A, Iinvention, particular when taking into consideration the temperaturestability of the polypeptides of this invention.

In a preferred embodiment, the polypeptide having cellobiohydrolaseactivity comprises an amino acid sequence having at least 85% sequenceidentity to SEQ ID NO: 12. wherein the polypeptide has the amino acidsequence of SEQ ID NO: 12 wherein one or more of the following aminoacid residues are modified by substitution or deletion: Q1. S2. P12.T15. S21, G23. T26. Q28. T29. G30. V32. N37. W40. T48. C50. N54. L60.E65. K69. V84. S90. D114, E119. F120. T121. L122. L123. D132. V133.G142. S148. M149. V155. Y158. N161. T162, K166. G170. Q175. F182. I183.G191. I203. D214. I215. A224. T231. G234. I237. S248, G254. W263. G269.L282. T285. G298. Y303. N307. G308. T310. E317. L318. S322. N324, G340.S341. D345. S357. M360. V363. D369. A372. P382. S388. T389. P390. T399.S400, Q406. N413. F423. P425. I426. G427. T429. P432. G435. N436. P437.G439. N441. R442, T444. T445. T446. T447. R449. P450. A451. T452. T453.S456, 5457. P458. G463. P464, S467. H459. C468. G470. G472. S474. P476.V478. C479, 5481. G482. T484. V487. L488, N489. Y491. Y492. Q494. C495.L496

Another aspect of the invention relates to the application of theisolated polypeptides and variants thereof of the present invention forthe complete or partial hydrolysis of cellulosic material. Thecellulosic material can be of natural, processed or artificial nature.“Cellulosic material” herein shall be defined as all sorts of pure,non-pure, mixed, blended or otherwise composed material containing atleast a fraction of β-1-4-linked D-glucosyl polymers of at least 7consecutive subunits. Prominent examples of cellulosic materials are allsort of cellulose containing plant materials like wood (soft and hard),straw, grains, elephant grass, hey, leaves, cotton and materialsprocessed there from or waste streams derived from such processes.Cellulosic material used in an enzymatic reaction is herein alsoreferred to as cellulosic substrate.

The hydrolysis of the cellulose material can be a sequential processfollowing cellobiohydrolase production or contemporary to the productionin the yeast cell (consolidated bioprocess). The expression ofcellulolytic enzymes in yeast is of special interest due to the abilityof many yeasts to ferment the released sugars (C6 or C5) to ethanol orother metabolites of interest.

A further embodiment of the invention thus relates to the application ofwhole cells expressing the polypeptide or variant thereof according tothe present invention for the processing of cellulosic materials.

In a particular embodiment, the present invention discloses the use of apolypeptide and variants thereof or the composition of the presentinvention for the enzymatic degradation of cellulosic material,preferably lignocellulosic biomass, and/or for textiles processingand/or as ingredient in detergents and/or as ingredient in food or feedcompositions.

EXAMPLES Example 1 Preparation of Pichia pastoris Expression Plasmid

Expression plasmids for the constitutive expression of protein fromtransformed Pichia pastoris hosts are prepared by assembly of anexpression cassette consisting of a Pichia pastoris gyceraldehydephosphate dehydrogenase (GAP) promoter, a Saccharomyces cerevisiae SPα(mating factor alpha signal peptide), a multiple cloning site (MCS) andthe 3′-GAP-terminator sequence. For selection purposes a kanamycineresistance gene is used under control of the EM7 or TEF promoter forbacterial or yeast selection purposes, respectively. The resultingplasmid vectors are designated as pV1 (FIG. 1) and pV2 (alternative MCS)Transformation and expression cultivation are done essentially asdescribed by Waterham, H. R., Digan, M. E., Koutz, P. J., Lair, S. V.,Cregg, J. M. (1997). Isolation of the Pichia pastorisglyceraldehyde-3-phosphate dehydrogenase gene and regulation and use ofits promoter. Gene, 186, 37-44 and Cregg, J. M.: Pichia Protocols inMethods in Molecular Biology, Second Edition, Humana Press, Totowa N.J.2007.

Example 2 Construction of Pichia pastoris Expression Constructs for CBHISequences

CBHI genes of Trichoderma viride (CBH-f), Humicola grisea (CBH-d),Thermoascus aurantiacus (CBH-e), Talaromyces emersonii (CBH-b), andfusions of the cellulose binding domain of Trichoderma reesei CBHI withthe Talaromyces emersonii CBHI (CBH-a) or the Humicola grisea CBHI(CBH-g) are amplified using the oligo nucleotide pairs and templates(obtained by gene synthesis) as given in the Table 4. The fusion geneencoding Seq ID. NO. 2 is generated by overlap extension PCR using thePCR-Fragments generated from SeqID NO. 5 and 11. Phusion DNA polymerase(Finnzymes) is used for the amplification PCR.

TABLE 4 Primers and templates for the amplification of CBH-a, CBH-b,CBH-d, CBH-e, CBH-f and CBH-g Fragment Primer forward Primer reverseTemplete CBH-f Trichoderma GAGGCGGAAGCACCC GGAGACGCAGAGCC SEQ IDviride CBHI TCTcaatctgcttgcaccttgca Cttattacaggcactgcgagt NO. 13gtc (SEQ ID NO: 20) agt (SEQ ID NO: 21) CBH-d Humicola griseaGAGGCGGAAGCACCC GGAGACGCAGAGCC SEQ ID GBHI TCTcagcaggctggtactattaCttacacgttcacggtagaac NO. 7 ctgc (SEQ ID NO: 22)cgattgggc (SEQ ID NO: 23) CBH-e Thermoascus GAGGCGGAAGCACCCGGAGACGCAGAGCC SEQ ID aurantiacus TCTcacgaggccggtaccgtaCTTAttagttggcggtgaag NO. 9 CBHI accgc (SEQ ID NO: 24)gtcgagt (SEQ ID NO: 25) CBH-b Talaromyces GAGGCGGAAGCACCC GGAGACGCAGAGCCSEQ ID emersonii CBHI TCTcagcaggccggcacggc CTTAtcacgaagcggtgaa NO. 5gacggc (SEQ ID NO: 26) ggtcgagt (SEQ ID NO: 27) CBH-a TalaromycesGAGGCGGAAGCACCC ATTACCTGTGCTACC SEQ ID part1 emersonii CBHTCTcagcaggccggcacggc gatcggaccaaacttaatgttc NO. 5 fusion fragment gacggcg (SEQ ID NO: 29) part 1 (SEQ ID NO: 28) CBH-a TrichodermaAAGTTTGGTCCGATCg GGAGACGCAGAGCC SEQ ID part2 reesei CBHIgtagcacaggtaatccttcagg CTTATTAtagacactgtga NO. 11 binding domain(SEQ ID NO: 30) gtagtaagggt fusion fragment (SEQ ID NO: 31) part2 CBH-aTalaromyces GAGGCGGAAGCACCC GGAGACGCAGAGCC 5a + 5b emersonii CBHITCTcagcaggccggcacggc CTTAtcattaatggtggtggt SEQ ID fusion proteingacggc (SEQ ID NO: 32) gatgatgag (SEQ ID NO: 33) NO. 2 CBH-gHumicola grisea aggcggaagcatgctcgcagc ggattacctgttaagcttccaat SEQ IDpart1 CBHI fusion aggctggtacaattactgc tggtccgaatctgatgtt NO. 19fragment part1 (SEQ ID NO: 34) (SEQ ID NO: 35) CBH-g Trichodermaaccaattggaagcttaacaggta atcttgcaggtcgacttatcatt SEQ ID part2 reesei CBHIatccttcaggtggtaatcc aatgatgatgatgatggtgtgc NO. 11 binding domain(SEQ ID NO: 36) a (SEQ ID NO: 37) fusion fragment part2 CBH-gHumicula grisea aggcggaagcatgctcgcagc atcttgcaggtcgacttatcatt 6a + 6bfusion protein aggctggtacaattactgc aatgatgatgatgatggtgtgc SEQ ID(SEQ ID NO: 38) a (SEQ ID NO: 39) NO. 15

PCR fragments of expected length are purified from agarose gels afterelectrophoresis using the Promega® SV PCR and Gel Purification kit.Concentration of DNA fragments are measured on a Spectrophotometer and0.2 pmol of fragments are treated with 9U of T4-DNA polymerase in thepresence of 2.5 mM dATP for 37.5 min at 22.5° C. and treated fragmentsare annealed with T4-DNA-Polymerase/dTTP treated SmaI-linearized pV1plasmid DNA and afterwards transformed into chemically competentEscherichia coli Top10 cells. Deviant from the described procedure theproduct generated by the primer pair according to the table lane 11encoding the Humicula grisea fusion protein fragments are cloned via theintroduced SphI and SafI site to pV2. Transformants are controlled bysequencing of isolated plasmid DNA.

Example 3 Expression of CBHI Genes in Pichia pastoris

Plasmids of Example 2 are transformed to electro-competent Pichiapastoris CBS 7435 cells and transformants are used to inoculate culturesin YPD medium containing 200 mg/l, which are incubated for 5 days at 27°C. in a rotary shaker at 250 rpm. Culture supernatants were separated bycentrifugation at 5000×g for 30 minutes in a Sorvall Avant centrifuge.Supernatants were concentrated on spin columns with cut-off size of 10kDa. Protein pattern of such concentrated supernatants were analyzed bySDS-PAGE (Laemmli et al.) and gels were stained with colloidal Commassieblue stain. Enzymatic activity was determined by incubation of thesupernatant with 2 mM solutions of p-nitrophenyl-β-D-lactoside or 200 μMsolutions of 4-methyl-umbelliferyl-β-D-lactoside in 50 mM sodium acetatebuffer (pH 5) for 1 hour. The reaction was stopped my addition of equalvolumes of 1 M sodium carbonate solution and determination of releasedp-nitrophenol or 4-methyl umbelliferone by measurement of the absorbanceat 405 nm or the fluorescence at 360 nm/450 nm excitation/emission.

Example 4 Genome Integration and Expression of the Talaromyces emersoniiCBHI-T. reesei CBHII-CBD Fusion Sequence in Pichia pastoris

TABLE 5 Fragment Primer forward Primer reverse Template Talaromyces GAGGCGGAAGCACCCTCTc ATTACCTGTGCTACCg SEQ ID emersonii CBH agcaggccggcacggcgacggc atcggaccaaacttaatgttcg NO. 5 fusion fragment(SEQ ID NO: 26) (SEQ ID NO: 29) part1 Trichoderma reeseiAAGTTTGGTCCGATCggtagc GGAGACGCAGAGCC SEQ ID CBHI binding acaggtaatccttcagg Cttatcattaatggtggtggtgat NO. 11 domain fusion (SEQ ID NO: 30) gatgag (SEQ ID NO: 33) fragment with 6x His-Tag part2Talaromyces  GAGGCGGAAGCACCCTCTc GGAGACGCAGAGCC OE 1a + 1b:emersonii CBHI  agcaggccggcacggcgacggc Cttatcattaatggtggtggtgat SEQ IDfusion protein (SEQ ID NO: 26) gatgag (SEQ ID NO: 33) NO. 17with 6x His-Tag part2

The DNA-fragment of the fusion gene are generated by 2 step overlapextension PCR using the oligo nucleotide pairs and synthetic templatesas indicated in the Table 4 (of Example 2). T4-DNA polymerase treatedfull length fragment was annealed with the linear pV3 vector fragment byslowly reducing the temperature from 75° C. to 4° C. The pV3 plasmidcontains a fusion of the mating factor alpha signal peptide to amultiple cloning site, situated downstream the of a Pichia pastoris AOXIpromoter. Transformation of the annealed solution into chemicalcompetent E. coli cells yields transformants, which are selected bytheir Teocine resistance checked for containing expected constructplasmid by restriction analysis and sequencing. pV3-CBH-a plasmidpreparations are linearized with SacI and approximately 1 μg of linearDNA-fragments are transformed to Pichia pastoris electrocompetent cells.94 Transformants from YPD-Zeocin plates are afterwards checked forexpression by cultivation in 500 μl 96-deepwell Plate cultures inBMMY-medium containing 1% methanol and 0.5% methanol was fed every 24 hfor 5 days (350 rpm/27° C.; humidified orbital shaker with 2.5 cmamplitude. Supernatants are tested for activity on 4-MUL and clones withhighest expression levels are selected and again evaluated under sameconditions.

For fermentation in an Infors Multifors bioreactor the strain producingthe highest enzyme concentration is selected. A YPD-Zeocin (100g/l)pre-culture is chosen for inoculation of Mineral medium consisting ofphosphate-buffer, magnesium sulphate and chloride, trace elements/biotinand glycerol, with pH calibration using ammonia and phosphoric acid.After metabolism of the batch glycerol (2%) additional glycerol feed ismaintained for 1 day before the feed is changed to methanol to shift toinductive conditions for the AOXI promoter. Under these conditions thefermentation is kept for 5 days. Cells are separated from thefermentation liquid by centrifugation at 5000×g for 30 minutes.Supernatants are analyzed for total Protein using Bradford Reagent andBSA Standards (Biorad). SDS-PAGE/Coomassie Brilliant blue staining isused to analyze the Protein Pattern on the SDS-PAGE.

Example 5 Trichoderma reesei Expression Vector Construct

SbfI/SwaI digested pSCMB100 plasmid DNA was transformed into Trichodermareesei SCF41 essentially as described by Penttilä et al 1997. 10 μg oflinear DNA was used for the transformation of 10⁷ protoplasts. Selectionof transformants was done by growth of the protoplasts on Mandel'sAndreotti media plates with overlay agar, containing hygromycine asselective agent (100 mg/l). Transformants were further purified bypassage over sporolation media plates and re-selection of spores onhygromycin media. From re-grown mycelia genomic DNA was isolated and thereplacement event verified by PCR. Transformants verified in being truereplacement strains were further tested for secretion of recombinantprotein.

Example 6 Expression of Talaromyces emersonii CBHI/Trichodermareesei-CBD Fusion (CBH-ah) Fusion with 6× His-Tag from Trichodermareesei

Expression of recombinant CBHI replacement strains of Talaromycesemersonii CBHI/Trichoderma reesei-CBD fusion with 6× His-Tag inTrichoderma reesei Q6A (ATCC 13631) was done in shakeflask culturescontaining 40 ml Mineral medium containing 2% Avicel in 300 ml flasksand cultivation at 30° C/250 rpm for 6 days. Supernatants recovered bycentrifugation and further analyzed by SDS-PAGE and Bradford Proteinassays.

Example 7 Screening Thermo Stability Variants

Random mutagenesis libraries of the Talaromyces emersoniiCBHI/Trichoderma reesei-CBD fusion (with 6× His-Tag) gene were generatedusing error prone PCR applying manganese containing bufferers andinbalanced dNTP concentrations in the Taq-DNA polymerase reactionmixture, used for PCR-amplification, essentially as described by Craigand Joyce (R. Craig Cadwell and G. F. Joyce, 1995. Mutagenic PCR, in PCRPrimer: a laboratory manual, ed. C. W. Dieffenbach and G. S. Dveksier,Cold Spring Harbor Press, Cold Spring Harbor, Me., 583-589). As templatethe wild type fusion gene (SeqID. NO. 17) or mutants thereof werechosen. Mutated PCR-Fragments were cloned to the pPKGMe Plasmid usingSphI and HindIII endonucleases and T4-DNA-ligase.

Libraries of the Talaromyces emersonii CBHI/Trichoderma reesei-CBDfusion (with 6× His-Tag) gene variants were distributed in 1536 wellplates with well occupation number close to 1. Enzyme was expressed over7 days in a volume of 4 μl YPG-G418 medium. For evaluation of theproperties of the variants 2 μl samples of culture supernatants weretransferred to plates containing a suspension of milled straw, acetatebuffer and beta-glucosidase. After incubation of the sealed reactionplates for 48 hours at defined temperatures the glucose concentrationwas determined using Amplex red in the presence of GOX and HRP byanalyzing the fluorescence level. Best-performing Hits werere-cultivated and re-evaluated. Plasmids of confirmed CBH-ah variantswere recovered (Pierce DNAzol Yeast genomic DNA Kit) and sequenced usingoligonucleotides alpha-f (5′ TACTATTGCCAGCATTGCTGC-3′,SEQ ID NO: 40) andoli740 (5′-TCAGCTATTTCACATACAAATCG-3′ SEQ ID NO: 41).

Example 8 Determination of Substrate Conversion Capacity at DifferentTemperatures for Indication of the Thermostability of CBH-ah-VariantsUsing 4-methylumbellifery-β-D-lactoside (4-MUL)

For precise comparison of the thermal stability culture supernatantscontaining the secreted cellobiohydrolase variants were diluted tenfoldin sodium acetate buffer (50 mM, pH 5) and 10 μl samples were incubatedwith 90 μl of 200 μM 4-MUL (in buffer) in the temperature gradient of anEppendorff Gradient Thermocycler. A temperature gradient of 20° C.reaching from 55° C. to 75° C. was applied to 12 reaction mixtures foreach sample for one hour. The temperature profile could be recordedafter addition of 100 μl 1M sodium carbonate solution to each reactionand measurement of the fluorescence intensity at 360 nm/454 nm in aTecan Infinite M200 plate reader. For comparison of the thermostabilitythe values were normalized between 1 and 0 for the maximum and minimumfluorescence count (FIG. 7).

TABLE 6 Listing of Mutants of SeqID NO. 18 with improved IT50 values.IT50-value (4- Mutations with respect to Seq. ID NO. 18 MUL) 1 wt 60.4+/− 0.6 2 R446G 60.6 3 Q1L, G4C, Q28R, A72C, S86T, E183M, D202V, S311N,D320N, T335I, 60.7 D346E, Q349R, P442S, N445D, R446G, H468Q 4 D346G,R453G 60.9 5 Y496F 60.9 6 T335I, D346A, T393A, D410G 61 7 T243I, T325A,V482A 61 8 N194R, T243R, Y374D, A375D 61.1 9 Q1L, G4C, Q28R, W40R, E65V,A72C, S86T, E183K, S192I, D202I, 61.1 H203R, F260C, S311N, D320N, T335I,D346G, Q349K, T392M, P442S, R446G, H468L, V482A 10 E65K 61.1 11 T48A61.2 12 G4C, A72C, T344M 61.2 13 T243R, A375D 61.3 14 W40R, K159E, N445D61.3 15 T344M 61.3 16 W40R, M234K 61.3 17 Q349R, T393A, P436S, N445D61.4 18 Q349R, A354T, D373E, N445D 61.4 19 G4C, W40R, A72C, T344M 61.420 G4C, A72C, N194L, T243Y, Q349R, Y374R, A375L 61.5 21 G4C, D64N, A72C,Q349K 61.5 22 Q349R, N445D 61.5 +/− 0.1 23 W40R, T344M 61.5 24 W40R,D346A, T393A 61.6 25 N158D, G486S, Y495C 61.6 26 D320E 61.6 27 A72V 61.628 E183V 61.6 29 W40R, C489R 61.7 30 A72V, T335I, D346A, T393A, N445D61.7 31 G4C, W40R, A72C, V313I, Q349R 61.8 32 W40R, Q349K 61.9 33 Q1L,G4C, T26N, A72C, D181N, E183K, Q349R 61.9 34 G4C, A72C, Q349K, E65V,Q349R 61.9 35 S311N 62 36 D320V, D346A, Q349R, T393A, N445D 62.1 37T335I 62.2 38 A72V, D346A 62.2 39 D320V, D346A, T393A, N445D 62.2 40G4C, A72C, N194S, T243W, Q349R, Y374S, A375G 62.3 41 E65V, Q349R 62.3 42Q1L, G4C, A72C, F179I, D181N, E183K, L290H, S301C, Q349R, 62.3 Q361R,D390G, G474S, Q498K 43 A72V, T335I, D346A, N445D 62.3 44 G4C, A72C,N194L, T243Q, Q349R, Y374P, A375V 62.3 45 Q1L, G4C, G23D, A72C, D111E,I116V, K118A, D181N, E183K, 62.3 V212L, Q349R, Q362G 46 G4C, A72C, D346G62.3 47 W40R, T344M, Q349K 62.3 48 G4C, Y163C 62.4 49 G4C, V32G, N49S,A72C, S193L, Q349R 62.4 50 A72V, Q349R, N445D 62.4 +/− 0.4 51 G4C, A72C,D346G, Q349R 62.5 +/− 0.1 52 Q1L, G4C, A72C, D181N, E183K, I321N, Q349R62.6 53 W40R, D320V, Q349K, P436S, N445D 62.7 54 G4C, A72C, D181N, Q349R62.7 55 G4C, A72C, Q349K 62.7 56 G4C, A72C, T344M, Q349K 62.8 57 G4C,A72C, T344M, D346G, Q349R 62.8 +/− 0.2 58 G4C, A72C, D320V, Q349K 62.859 G4C, A72C, P224L, F306Y, Q349R 62.9 60 G4C, A72C 62.9 +/− 0.6 61A72V, D346A, T393A 63 62 G4C, A72C, Q349R, R446S, T456A 63 63 G4C, W40R,A72C, T344M, Q349K 63 64 A72V, D320V, D346A 63.1 65 G4C, A72C, N194Y,T243L, Q349R, Y374S, A375R 63.1 66 G4C, A72C, Q349K, T448A, T449A 63.267 G4C, E65V, A72C, Y244H, Q349R 63.3 68 G4C, A72C, D202G, D320N, Q349R,A358E 63.4 69 G4C, A72C, D320V, Q349R 63.4 70 G4C, A72C, Q349K, S86T63.4 71 A72V, T335I, D346A, T393A, P436S 63.4 72 G4C, A72C, E183V,K304R, Q349K 63.5 73 G4C, A72C, T243G, Q349R, Y374P, A375M 63.5 74 G4C,A72C, Q349R, T465I 63.6 75 G4C, A72C, Q349R 63.6 +/− 0.5 76 G4C, A72C,N194V, T243M, Q349R, Y374A, A375T 63.6 77 G4C, D64N, A72C, Q349R, A358E,P464Q 63.6 78 G4C, A72C, Q349K, Q28R, S193T, Q490L 63.6 79 G4C, A72C,E183K, Q349K 63.6 80 G4C, A72C, S311N, Q349K, A455T 63.6 81 G4C, A72C,N194K, Q349R, Y374P, A375Q 63.6 82 G4C, A72C, D181N, Q349K 63.6 83 W40R,D320V, Q349K, T393A, N445D 63.7 84 W40R, T335I, D346A, T393A 63.7 85Q1L, G4C, A72C, D181N, E183K, N327Y, Q349R 63.7 86 A72C, L119L, T335I,Q349R, G486D 63.7 87 G4C, A72C, N194K, T243P, Q349R, Y374H, A375E 63.788 G4C, A72V, Q349R, P462del 63.8 89 G4C, A72C, S236Y, Q349R 63.8 90G4C, A72C, S311G, Q349K 63.8 91 A72V, D320V, T335I, D346A, T393A, N445D63.8 92 G4C, A72C, S86T, M234V, Q349K 63.8 93 Insertion at positionK159(CGRNK)undT457(AAATT), Q1L, G4C, 63.9 Q28R, E65V, A72C, D181N,E183M, P224L, S311G, D320I, D346E, Q349K, T393V, Y422F, P442S, N445D,R446G, H468L, V482I 94 G4C, A72C, G251R, Q349R 63.9 95 G4C, A72C, Q349K,D320V 63.9 +/− 0.7 96 A72V, T335I, D346A, T393A 63.9 97 G4C, A72C,E183K, Q349R 63.9 98 Q1L, G4C, A72C, H203R, Q349K, P442S 63.9 99 G4C,A72C, Q349K, G434S, G470D 64 100 G4C, W40R, A72C, Q349K 64 101 G4C,A72C, Q349R, V367A 64 102 Q1L, G4C, A6V, C50S, A72C, I180N, D181N,E183K, Q349R, T457P, 64 C472R, C499G 103 G4C, A72C, S311G, D320V, Q349K64 104 W40R, T335I, D346A, T393A, P436S 64 105 Q1L, G4C, A72C, D181N,E183K, T243S, Q349R, P386S 64.1 106 A72V, D346A, T393A, N445D 64.1 107Q1L, G4C, A72C, K154R, Q349K, T393I 64.1 108 G4C, A72C, N194G, T243F,Q349R, Y374P, A375R 64.1 109 A72V, D320V, D346A, T393A, N445D 64.1 110A72C, L119L, Q172Q, Q349K, T488I 64.1 111 G4C, A72C, E183V, Q349K 64.1112 G4C, A72C, E183K, N318Y, Q349K 64.1 113 W40R, A221V, T449A, C483R64.1 114 G4C, A72C, K92R, Q349K, N493D 64.1 115 M234I, G438del 64.2 116Q1L, G4C, A72C, S90T, D181N, E183K, Q349R 64.2 117 G4C, A72C, Q349R,G459D 64.2 118 G4C, A72C, Q349R, Y422F 64.2 119 G4C, T48A, A72C, Q349R,P480S 64.2 120 E187K, D320V, P442del 64.2 121 G4C, S24N, E65K, A72C,Q349R, I430L, G439D 64.2 122 A72V, D320V, T335I, D346A, T393A, P436S64.3 123 Q1L, G4C, A72C, S193P, Q349K, V482I 64.3 124 G4C, A72C, D320V,Q349K, G443D, L492Q 64.3 125 Q1L, G4C, A72C, DV152-K159, D181N, E183K,Q349R 64.4 126 Q1L, G4C, A72C, Q349K 64.4 +/− 0.5 127 Q1L, G4C, A72C,D181N, E183K, M234L, V313I, Q349R, H468R 64.4 128 Q1L, G4C, A72C, D181N,E183K, I200N, Q349R 64.4 129 G4C, A72C, N194K, T243Y, Q349R, A375N 64.4130 Q1L, G4C, Q28R, A72C, Q349K, H468L 64.5 131 G4C, E65V, A72C, Q349R64.5 132 D320V, Q349K 64.5 133 Q1L, G4C, A72C, S311G, Q349K, H468R 64.5134 G4C, A72C, T243Q, Q349R, Y374P, A375M 64.5 135 Q1L, G4C, A72C,D320V, Q349R 64.5 136 Q1L, G4C, T15S, A72C, Y244F, Q349K 64.5 137 G4C,A72C, E183K, D346E, Q349R 64.6 138 T243C, A375C 64.6 139 Q1L, G4C, A72C,Q349K, T392M 64.6 140 G4C, A72C, D202N, S311N, Q349R, N493D 64.6 141G4C, A72C, N194D, T243A, Q349R, Y374P, A375Y 64.6 142 G4C, A72C, N194Y,T243V, Q349R, Y374P 64.7 143 Q1L, G4C, A72C, Q349R 64.7 144 G4C, Q28R,E65K, A72C, S86T, D202N, H203R, S311N, D320I, 64.7 A340G, D346A, Q349K,T393A, Y422F, P442S, R446S, H468L, V482A 145 G4C, A72C, D202N, Q349R64.7 146 G4C, A72C, P224L, Q349R 64.7 147 N194C, Y374C 64.7 148 Q1L,G4C, A72C, D181N, E183K, T229M, A340T, Q349R, V491I 64.7 149 Q1L, G4C,A72C, D181N, Q349R 64.8 150 G4C, A72C, D320V, D346V, Q349K 64.8 151 Q1L,G4C, A72C, V152A, Q349K 64.8 152 Q1L, G4C, Q28R, A72C, Q349K 64.8 153Q1L, G4C, A72C, Q349K, Y422F 64.8 154 G4C, A72C, D202V, D320V, Q349K64.8 155 Q1L, G4C, A72C, Y155S, D181N, E183K, Q349R 64.8 156 Q1L, G4C,A72C, D181N, D247N, Q349K 64.8 157 Q1L, G4C, A68T, A72C, Q349K, G439D,R453S 64.9 158 Q1L, G4C, A72C, Q349K, H468R 64.9 159 Q1L, G4C, D64N,A72C, Q349K 64.9 160 G4C, A72C, E183K, Q349R, P464L 64.9 161 G4C, A72C,D181N, P224L, Q349K   65 +/− 0.6 162 G4C, A72C, N194I, T243Y, Q349R,Y374P, A375R 65 163 Q1L, G4C, A72C, Q349K, P462L 65 164 Q1L, G4C, A72C,E183K, Q349R 65 165 G4C, A72C, S311G, Q349R 65 166 Q1L, G4C, A72C,S311N, Q349K, G463D 65.1 167 Q1L, G4C, A72C, S86T, Q349R 65.1 168 Q1L,G4C, A72C, D181N, E183K, G231D, Q349R 65.1 169 Q1L, G4C, A72C, S89N,D181N, E183K, Q349R 65.2 170 Q1L, G4C, E65K, A72C, Q349K 65.2 171 Q1L,Q2P, G4C, W40R, E65M, A72C, S86T, S192L, D202N, H203R, 65.2 S311D,D320I, T335I, D346G, Q349K, T392M, Y422F, R446G 172 Q1L, G4C, S24T,T26I, Q28R, N29Y, G30A, E65V, A68T, A72C, 65.2 Y155C, D181N, E183M,Q190K, P224L, S311G, D320I, D346E, Q349K, T393V, Y422F, P442S, N445D,R446G, H468L, V482I 173 G4C, A72C, E183K, D202Y, N310D, Q349R 65.3 +/−0.7 174 G4C, A72C, N194I, T243D, Q349R, Y374P, A375Y 65.3 175 Q1L, G4C,A72C, D181N, E183K, Q349R, T456I 65.3 176 G4C, Q28R, A72C, S86T, E183K,P224L, S311N, N318Y, T335I, 65.3 D346G, Q349R, T393I, P441A, P442S,R446G, H468L, V482I 177 Q1L, G4C, A72C, D181N, Q349K, T451S 65.3 +/− 0.1178 Q1L, G4C, A72C, D181N, E183K, T243I, N246D, Q349R, T488I 65.4 179Q1L, G4C, G23N, A72C, D110G, I116V, L119I, D181N, E183K, 65.4 D211G,D293R, N310D, Q349R, Q362R, G363P, M364S 180 G439V, N440E, P441S, P442Q65.4 181 G431D, S431V, T433E, G434S, N435Q 65.5 182 Q1L, G4C, Q28R,E65V, A68T, A72C, Y155C, D181N, E183M, P224L, 65.6 S311G, D320I, D346E,Q349K, T393V, Y422F, P442S, N445D, R446G, H468L, V482I 183 G4C, A72C,N194C, Y374C 65.6 184 Q1L, G4C, A72C, S86T, D181N, E183K, Q349R, T393S65.6 185 Q1L, G4C, A72C, D181N, E183K, Q349R, S485T 65.7 186 Q1L, G4C,G23N, A72C, V84A, D110G, D111H, I116E, F117Y, K118A, 65.7 D181N, E183K,D293R, T295S, Q349R, M364L 187 Q1L, G4C, A72C, D181N, E183K, Q349R,R453K 65.8 188 Q1L, G4C, A72C, A145T, H203R, Q349K, T403K 65.8 189 Q1L,G4C, A72C, D181N, E183K, Q349R, N445S 65.8 190 Q1L, G4C, A72C, D181N,E183K, M234I, Q349R 65.8 191 Q1L, G4C, A72C, D181N, E183K, Q349R, T465S65.9 192 Q1L, G4C, A72C, D181N, E183K, T297N, Q349R 66 193 Q1L, G4C,A72C, S311G, Q349K 66 194 G4C, Q28R, E65M, A72C, S86T, E183K, S192I,H203R, S311N, 66 D346E, Q349K, T392M, T393A, Y422F, N445D, R446S 195Q1L, G4C, A72C, D202N, Q349K, G486D 66 196 Q1L, G4C, A72C, S99T, D181N,E183K, Q349R, T450I 66 197 Q1L, G4C, A72C, I200F, Q349K, L500I 66 198Q1L, G4C, A72C, D181N, E183K, Q349R, G434S 66 199 Q1L, G4C, A31S, A72C,D181N, E183K, Q349R 66 200 Q1L, G4C, Q28L, A72C, D181N, E183K, Q349R 66201 Q1L, G4C, A72C, D181N, E183K, Q349R, P436S 66.1 202 Q1L, G4C, A72C,D181N, E183K, T233S, Q349R 66.1 203 Q1L, G4C, A72C, D202N, Q349K 66.1+/− 0.1 204 Q1L, G4C, A68T, A72C, Q349K 66.1 205 Q1L, G4C, A21T, A72C,D181N, E183K, Q349R, P454S 66.1 +/− 0.1 206 Q1L, G4C, A72C, D346V, Q349K66.2 207 Q1L, G4C, Y47F, A72C, D181N, E183K, Q349R, P436S, S461R 66.2208 Q1L, G4C, A72C, D181N, E183K, M234T, Q349R 66.3 209 Q1L, G4C, A72C,N157S, D181N, E183K, Q349R 66.3 210 Q1L, G4C, A72C, D181N, E183K, Q349R66.3 +/− 0.4 211 As1-72ausT. reesei, D181N, P224L, S311D, N318Y, D320N,D346A, 66.3 Q349K, T392M, T393I, Y422F, N445D, R446G, H468Q, V482I 212G4C, A72C, N194Q, T243V, Q349R, Y374P, A375Y 66.3 213 Q1L, G4C, A72C,D181N, E183K, I314F, Q349R, N445D 66.3 214 Q1L, G4C, A72C, Q349K, T392K66.3 215 Q1L, G4C, A72C, D181N, E183K, Q349R, T451A 66.4 216 Q1L, G4C,A72C, D181N, E183K, M234V, Q349R 66.4 217 Q1L, G4C, A21S, A72C, D181N,E183K, Q349R 66.5 218 A21C, P429C 66.5 +/− 0.4 219 Q1L, G4C, A72C,D181N, E183K, Q349R, N493D 66.5 220 Q1L, G4C, A72C, S311N, Q349K 66.5221 Q1L, G4C, Q28R, E65V, A72C, D181N, E183V, D228N, S311N, 66.5 N318Y,D346E, Q349R, Y422F, P442S, N445D, R446G, H468L, V482T 222 G4C, A72C,N194C, Q349R, Y374C 66.5 223 Q1L, G4C, A72C, D181N, E183K, Q349R, A455V66.5 +/− 0.3 224 Q1L, G4C, A72C, D181N, E183K, Q349R, T400S 66.6 225Q1L, G4C, T26I, A72C, D181N, E183K, Q349R 66.6 +/− 0 226 Q1L, G4C, A72C,D181N, E183K, N310D, Q349R, T392S, G463D 66.7 227 Q1L, G4C, A72C, D129N,D181N, E183K, Q190L, G266S, I276V, 66.7 +/− 0.2 Q349R, P386L, F427Y 228Q1L, G4C, A72C, D181N, E183K, D202N, Q349R 66.7 229 Q1L, G4C, A72C,Y155C, D181N, E183K, Q349R 66.8 230 Insertion at position P454(ATAAA),Q1L, G4C, Q28R, E65V, A72C, 66.8 D181N, E183M, P224L, S311G, D320I,D346E, Q349K, T393V, Y422F, P442S, N445D, R446G, H468L, V482I 231Insertion at position K159(CGRNK), Q1L, G4C, Q28R, E65V, A72C, 66.8D181N, E183M, P224L, S311G, D320I, D346E, Q349K, T393V, Y422F, P442S,N445D, R446G, H468L, V482I 232 Q1L, G4C, A72C, D181N, E183K, N246K,Q349R   67 +/− 0.6 233 G4C, W40R, E65V, A72C, S86T, D181N, E183K, D202I,H203R,   67 +/− 0.5 S311D, D320N, D346V, Q349R, T392M, T393A, Y422F,P442S, H468Q, V482A 234 Q1L, G4C, A72C, Y155C, Q349K   67 +/− 0.4 235Q1L, G4C, A68T, A72C, D181N, E183K, Q349R   67 +/− 0.3 236 Q1L, G4C,S24T, T26I, Q28R, N29Y, G30A, Y47F, E65V, A68T, A72C, 67.3 Y155C, D181N,E183M, Q190K, P224L, T229M, G231D, M234T, S311G, D320I, A340S, D346E,Q349K, D390E, T393V, Y422F, P442S, N445D, R446G, T448A, R453G, H468L,P480S, V482I 237 Q1L, G4C, W40R, E65M, A72C, S86T, S192L, D202N, H203R,S311D, 67.3 D320I, T335I, D346G, Q349K, T392M, Y422F, R446G 238 Q1L,G4C, A72C, S86T, D181N, E183K, D320V, Q349R 67.3 +/− 0.4 239 G4C, Q28K,A72C, S86T, E183M, D202N, P224L, S311G, N318Y, 67.4 D320N, D346A, Q349R,T392M, T393I, P442S, H468L, V482I 240 Q1L, Q2P, G4C, Q28R, W40R, E65K,A72C, D181N, S192L, D202I, 67.4 H203R, P224L, S311G, D320I, D343A,D346A, Q349K, P442S, N445D, R446G, V482A 241 Q1L, G4C, Q28R, E65K, A72C,E183M, D202I, P224L, D320N, D346V, 67.4 Q349K, T392M, T393V, Y422F,N445D, R446G, H468L, V482T 242 Q1L, G4C, Q28R, E65V, A72C, S86T, E183K,D202V, S311G, N318Y, 67.4 D320I, D346G, Q349K, T393V, Y422F, N445D,R446S, H468Q, V482T 243 Q1L, G4C, W40R, E65M, A72C, D181N, E183K, S192P,D202N, 67.4 P224L, S311D, N318Y, D320V, D346G, Q349K, T392M, N445D,R446G, H468L, V482T 244 Q1L, G4C, E65V, A72C, D181N, E183K, P224L,S311G, D320N, 67.6 D346G, Q349R, T392M, T393I, R446G, H468L, V482I 245Insertion at position G151(CGRSG), Q1L, G4C, Q28R, E65V, A72C, 67.6D181N, E183M, P224L, S311G, D320I, D346E, Q349K, T393V, Y422F, P442S,N445D, R446G, H468L, V482I 246 Q1L, G4C, E65M, A72C, D181N, E183M,D202Y, P224L, S311D, 67.6 N318Y, D320I, T335I, D346A, Q349K, T392M,T393I, N445D, R446G, T448A, H468Q, V482A 247 Q1L, G4C, G23A, A72C,D110S, D111H, I116V, F117Y, K118A, 67.6 L120M, D181N, E183K, D293H,G294A, N310E, Q349R, Q362G, M364S 248 Q1L, G4C, A72C, D181N, E183K,Q349R, T421I, G439D 67.7 249 G4C, Q28K, E65M, A72C, S86T, V152A, D181N,E183V, S192L, 67.7 +/− 0.3 D202N, S311N, D320N, D346E, Q349R, T387A,T392M, T393I, Y422F, P442S, R446S, H468L, G476D, V482I 250 Q1L, G4C,W40R, E65V, A72C, S86T, E183V, G188C, S192T, D202Y, 67.7 H203R, P224L,S311N, D320V, D346E, Q349K, T393V, Y422F, N445D, R446G, H468Q, V482I 251Q1L, G4C, E65M, A72C, S86T, E183M, D202N, P224L, T335I, D346G, 67.7Q349K, T392M, T393A, P442S, N445D, R446G, H468Q, V482A 252 Q1L, G4C,Q28R, E65V, A72C, D181N, E183M, P224L, S311G, D320I, 67.8 A340S, D346E,Q349K, D390E, T393V, Y422F, P442S, N445D, R446G, H468L, P480S, V482I 253Q1L, G4C, W40R, E65K, A72C, S86T, E183K, S192L, D202Y, P224L, 67.9 +/−0.4 D320I, D346E, Q349R, P442S, R446G, H468R, V482T 254 Q1L, G4C, A21T,T26I, Q28R, E65V, A68T, A72C, Y155C, D181N, 67.9 E183M, Q190K, D202N,P224L, S311G, N318Y, D320I, D346E, Q349K, T393V, Y422F, N445D, R446G,R453G, H468Q, P480S, V482I 255 Q1L, G4C, Q28R, E65V, A72C, S86T, D181N,D202N, P224L, S311G, 68 N318Y, D320I, T335I, D346E, Q349K, T393V, Y422F,N445D, R446G, H468Q, V482I 256 Insertion at position P464(THAAA), Q1L,G4C, Q28R, E65V, A72C, 68 D181N, E183M, P224L, S311G, D320I, D346E,Q349K, T393V, Y422F, P442S, N445D, R446G, H468L, V482I 257 Q1L, G4C,Q28R, E65V, A72C, E183M, S311G, D346E, Q349K, 68 T393V, Y422F, P442S,N445D, R446G, H468L, V482I 258 Q1L, G4C, T7Q, A8S, N10T, S24T, T27Q,Q28R, N29T, V41T, G46S, 68 Y47S, T52D, D57S, D64N, E65V, Q69K, A72C,D181N, P224L, S311D, N318Y, D320N, D346A, Q349K, T392M, T393I, Y422F,N445D, R446G, H468Q, V482I 259 Insertion at positionK159(CGRNK)undT457(AAATT), Q1L, G4C, 68.1 +/− 1.9 Q28R, E65V, A72C,E183M, P224L, S311G, D320I, D346E, Q349K, T393V, Y422F, P442S, N445D,R446G, H468L, V482I 260 Q1L, G4C, Q28K, W40R, E65V, A72C, D181N, E183V,S192P, D202V, 68.1 H203R, S311G, D320N, D346E, Q349K, T392M, T393A,Y422F, V482I 261 Q1L, G4C, Q28R, E65V, A72C, G139S, D181N, E183M, P224L,68.1 S311G, D320I, D346E, Q349K, T393V, Y422F, P442S, N445D, R446G,H468L, V482I 262 Q1L, G4C, Q28R, E65V, A72C, S86T, D181N, E183M, D202N,P224L, 68.2 S311G, N318Y, T335I, D346E, Q349K, T393V, Y422F, N445D,R446G, H468L, V482I 263 Q1L, G4C, Q28R, E65K, A72C, D181N, S311N, N318Y,D346E, 68.2 +/− 0.7 Q349K, T393V, Y422F, N445D, R446G, H468Q, V482I 264Q1L, G4C, W40R, A72C, S192L, D202N, H203R, P224L, S311N, 68.2 +/− 0D320I, T335I, D346V, Q349R, T393I, N445D, R446G, H468Q, V482T 265 Q1L,G4C, Q28K, E65K, A72C, E183M, D202N, P224L, T229S, 68.2 S311G, D320I,T335I, D346V, Q349R, T393V, H468Q, V482A 266 Q1L, G4C, Q28R, G30A, E65M,A72C, D181N, D202N, P224L, S311D, 68.2 N318Y, D346E, Q349K, T392M,T393V, Y422F, P442S, N445D, R446S 267 Q1L, G4C, Q28R, E65V, A72C, S68T,E183M, S311N, T335I, D346E, 68.2 Q349K, T393V, Y422F, N445D, R446G,H468L, V482T 268 Q1L, G4C, Q28K, E65K, A72C, D181N, D202N, S311N, N318Y,T335I, 68.3 D346E, Q349K, T393V, Y422F, P442S, N445D, R446G, H468L,V482I 269 T243C, A375C, N194C, Y374C 68.3 270 Q1L, G4C, Q28K, E65K,A72C, S86T, E183M, S311G, D346V, Q349R, 68.3 T392M, T393V, Y422F, P442S,N445D, R446G, H468L, S478Y, V482I 271 Q1L, G4C, Q28R, E65V, A72C, D181N,E183M, S311G, D346E, 68.3 Q349K, T393V, Y422F, P442S, N445D, R446G,H468L, V482I 272 Q1L, G4C, Q28R, E65V, A72C, S86T, D181N, D202N, S311N,N318Y, 68.4 D320I, T335I, D346E, Q349K, T393I, Y422F, N445D, R446G,H468Q, V482I 273 Q1L, G4C, Q28R, E65K, A72C, S86T, E183K, D202N, P224L,S311G, 68.4 T335I, D346A, Q349K, T393V, Y422F, N445D, R446G, H468L,V482T 274 Q1L, G4C, Q28R, E65V, A72C, L120P, D181N, E183M, P224L, 68.4S311G, D320I, D346E, Q349K, T393V, Y422F, P442S, N445D, R446G, H468L,V482I 275 Q1L, G4C, Q28K, E65V, A72C, S86T, E183M, S311N, N318H, D320V,68.4 D346V, Q349K, T392M, T393A, R446S, H468L, V482I 276 Q1L, G4C, Q28K,E65V, A72C, D202N, H203R, S311G, T335I, D346V, 68.4 Q349K, T393A, P442S,R446G, H468L, V482I 277 M146C, G167C 68.5 278 Q1L, G4C, Q28R, E65V,A72C, E183M, S311G, D320I, D346E, 68.5 Q349K, T393V, Y422F, P442S,N445D, R446G, H468L, V482I 279 Q1L, G4C, Q28R, E65K, A72C, D181N, D202N,H203R, P224L, 68.5 S311D, D346G, Q349K, P442S, R446S, H468Q, V482T 280Q1L, G4C, Q28R, E65M, A72C, D181N, E183M, S311G, N318Y, 68.5 D320I,T335I, D346E, Q349K, D390E, T393V, Y422F, P442S, N445D, R446G, H468Q,V482T 281 Q1L, G4C, T27S, Q28R, E65V, Q69R, A72C, L120P, D181N, E183M,68.5 D202N, D247N, S311G, D346E, Q349K, K355Q, T387S, T393V, Y422F,N445D, R446G, T451S, G463V, H468Q, V482T, S485T 282 Q1L, G4C, Q28R,E65K, A72C, E183V, S192T, D202N, S311G 68.6 +/− 0.6 D320V, D346A, Q349K,P442S, N445D, R446G, H468R, V482A 283 Q1L, G4C, W40R, E65V, A72C, S86T,E183M, D202N, P224L, S311G, 68.6 D320V, D346E, Q349R, T393I, P442S,R446G, H468R, V482I 284 Q1L, G4C, Q28N, E65K, A72C, D181N, E183M, D202N,H203R, 68.6 S311G, N318Y, D320N, Q349R, T393V, Y422F, P442S, N445D,R446G, H468L, V482I 285 Q1L, G4C, Q28R, V41T, G46S, Y47S, T52D, E65V,A72C, D181N, 68.6 P224L, S311D, N318Y, D320N, D346A, Q349K, T392M,T393I, Y422F, N445D, R446G, H468Q, V482I 286 Q1L, G4C, Q28R, E65K, A72C,D181N, E183M, P224L, S311G, D320I, 68.6 D346E, Q349K, T393V, Y422F,P442S, N445D, R446G, H468L, V482I 287 Q1L, G4C, Q28R, W40R, A72C, S86T,D181N, S192T, D202N, P224L, 68.6 +/− 1 S311G, N318Y, D320V, D346E,Q349K, T392M, T393I, P442S, N445D, R446G, H468Q, V482I 288 Q1L, G4C,A21T, T26I, Q28R, G30A, E65V, A68T, A72C, Y155C, 68.6 D181N, Q190K,D202N, P224L, T229M, S311G, N318Y, D320I, T335I, D346E, Q349K, T393V,Y422F, N445D, R446G, R453G, H468Q, V482I 289 Q1L, G4C, Q28K, W40R, A72C,S86T, D181N, E183M, S192I, D202Y, 68.6 +/− 0.4 T299S, S311N, N318Y,D320I, D346V, Q349R, T393I, P442S, H468L 290 Q1L, G4C, Q28R, E65V, A72C,D181N, E183M, P224L, T229M, 68.6 G231D, M234T, S311G, D320I, D346E,Q349K, T393V, Y422F, P442S, N445D, R446G, H468L, V482I 291 Q1L, G4C,S24T, T26I, Q28R, N29Y, G30A, E65V, A68T, A72C, 68.7 Y155C, D181N,E183M, Q190K, P224L, T229M, G231D, M234T, S311G, D320I, A340S, D346E,Q349K, D390E, T393V, Y422F, P442S, N445D, R446G, T448A, R453G, H468L,P480S, V482I 292 Q1L, G4C, Q28R, E65V, A72C, D181N, E183M, P224L, S311G,D320I, 68.7 D346E, Q349K, T393V, Y422F, P442S, N445D, R446G, G463V,H468L, V482I 293 Q1L, G4C, Q28R, E65K, A72C, E183M, P224L, S311G, D346E,68.7 Q349K, T393V, Y422F, P442S, N445D, R446G, H468L, V482I 294 Q1L,G4C, Q28R, E65V, A72C, S86T, D181N, E183M, D202N, P224L, 68.7 S311G,N318Y, D346E, Q349K, T393V, Y422F, P442S, N445D, R446G, H468L, V482T 295Q1L, G4C, Q28R, W40R, E65V, A72C, S86T, S192T, D202V, H203R, 68.7 S311N,N318Y, D346A, Q349K, T392M, T393A, Y422F, P442S, N445D, V482A 296Insertion at position K159(CGRNK), G434(AAATG)undT457(AAATT), 68.7 +/−1.1 Q1L, G4C, Q28R, E65V, A72C, E183M, P224L, S311G, D320I, D346E,Q349K, T393V, Y422F, P442S, N445D, R446G, H468L, V482I 297 Q1L, G4C,Q28K, E65K, A72C, D181N, H203R, P224L, S311G, 68.7 +/− 0 D320V, D346G,Q349K, T392M, T393I, P442S, N445D, R446G, H468L, V482A 298 Q1L, G4C,Q28R, E65K, A72C, E183M, S311G, D346E, Q349K, 68.8 T393V, Y422F, P442S,N445D, R446G, H468L, V482I 299 Q1L, G4C, Q28R, E65K, A72C, E183M, S311G,D346E, Q349K, 68.8 +/− 0.2 T393V, Y422F, P442S, N445D, R446G, H468L,V482I 300 Q1L, G4C, Q28R, E65V, A72C, E183K, D202I, P224L, S311G, D320I,68.8 Q349K, T393V, P442S, N445D, R446S, H468L, V482I 301 Q1L, G4C, Q28K,A72C, S86T, D181N, E183V, S192T, D202N, P224L, 68.8 S311G, N318Y, D320V,D346G, Q349R, T392M, Y422F, P442S, N445D, R446S, H468L, V482A 302 G4C,Q28R, E65K, A72C, S86T, D181N, E183M, S192L, D202N, 68.8 H203R, P224L,S311D, D346E, Q349R, T392M, T393A, Y422F, P442S, N445D, R446G, P462L,H468L, V482T 303 Q1L, G4C, W40R, E65K, A72C, Q109R, D181N, E183M, S192I,D202I, 68.8 +/− 0.2 H203R, S245T, D346A, Q349R, T393A, Y422F, P442S,N445D, R446G, H468R, V482A 304 Q1L, G4C, Q28K, E65M, A72C, D181N, D202N,S311N, T335I, 68.9 D346E, Q349K, T393I, Y422F, P442S, N445D, R446G,H468Q, V482I 305 Q1L, G4C, N10D, Q28R, E65V, Q69R, A72C, D181N, E183M,P224L, 68.9 S311G, D320I, D346E, Q349R, T393V, N417Y, Y422F, P442S,N445D, R446G, H468L, V482I 306 Q1L, G4C, Q28R, W40R, E65V, A72C, D202Y,H203R, P224L, T299I, 68.9 N318Y, D320V, D346A, Q349K, T392M, T393A,P442S, N445D, R446G, H468R, V482T 307 G4C, Q28R, E65V, A72C, E183M,D202N, P224L, S311G, N318Y, 68.9 D320V, D346G, Q349K, T392M, Y422F,R446G, H468Q 308 Q1L, G4C, Q28K, A72C, S86T, E183K, S311G, D320V, D346A,68.9 Q349R, T392M, T393I, Y422F, P442S, R446S, H468Q 309 Q1L, G4C, Q28R,E65M, A72C, D181N, P224L, S311N, D320N, T335I, 68.9 D346E, Q349K, T392M,Y422F, N445D, R446S, H468L, V482T 310 Q1L, G4C, Q28K, E65V, A72C, D181N,D202N, S311G, T335I, D346E, 68.9 Q349K, T393V, Y422F, N445D, R446G,H468Q, V482I 311 Q1L, G4C, Q28R, E65K, A72C, D181N, E183M, S311N, N318Y,T335I, 68.9 D346E, Q349K, T393V, Y422F, N445D, R446G, H468L, V482I 312Q1L, G4C, Q28R, E65K, A72C, E183M, S311G, D320I, D346E, 68.9 Q349K,T393V, Y422F, P442S, N445D, R446G, H468L, V482I 313 Q1L, G4C, Q28R,E65V, A72C, D181N, E183M, S311G, D320I, 69 D346E, Q349K, T393V, Y422F,P442S, N445D, R446G, H468L, V482I 314 Q1L, G4C, W40R, E65V, A72C, S86T,D181N, S192T, D202N, H203R,   69 +/− 0.3 P224L, S311G, N318Y, T335I,D346G, Q349K, T392M, T393V, Y422F, N445D, R446G, H468Q 315 Q1L, G4C,Q28R, E65K, A72C, E183M, S311N, D320I, D346E, 69 Q349K, T393I, Y422F,P442S, N445D, R446G, H468Q, V482T 316 Q1L, G4C, T7Q, A8S, N10T, Q28R,D57S, D64N, E65V, Q69K, A72C, 69 D181N, P224L, S311D, N318Y, D320N,D346A, Q349K, T392M, T393I, Y422F, N445D, R446G, H468Q, V482I 317 Q1L,G4C, Q28R, E65M, A72C, S86T, D181N, E183M, D202N, P224L,   69 +/− 0.9S311G, D346A, Q349K, T393I, Y422F, P442S, N445D, R446G, H468Q, V482T 318Q1L, G4C, N10D, Q28R, E65V, Q69R, A72C, K92R, K118Q, D181N, 69 E183M,D202N, T280A, S311G, T335I, D346E, Q349K, K355Q, T387S, T393V, D404N,Y422F, N445D, R446G, P462L, G463V, H468Q, V482I, S485T 319 Q1L, G4C,Q28R, E65M, A72C, E183M, D202N, S311G, D320I, T335I, 69 D346E, Q349K,T393I, Y422F, N445D, R446G, H468Q, V482I 320 Q1L, G4C, Q28K, E65K, A72C,D181N, E183M, D202N, S311D, 69 D320V, T335I, D346G, Q349K, T393I, Y422F,P442S, N445D, H468L, V482I 321 Q1L, G4C, Q28K, E65K, A72C, S86T, P224L,S311N, D320I, T335I, 69 D346E, Q349K, T393V, Y422F, N445D, R446G, H468Q,V482T 322 Q1L, G4C, Q28R, E65V, A72C, S86T, D181N, E183M, D202N, P224L,69 S311N, D320I, D346E, Q349K, T393V, Y422F, P442S, N445D, R446G, H468Q,V482T 323 G4C, E65K, A72C, S86T, E183M, D202I, P224L, S311N, N318Y, 69D320N, T335I, D346V, Q349R, T393V, Y422F, P442S, R446S, H468L 324 Q1L,G4C, Q28R, E65K, A72C, D202N, S311N, T335I, D346E, Q349K, 69 T393I,Y422F, P442S, N445D, R446G, H468L, V482T 325 Q1L, G4C, E65V, A72C,D181N, E183K, D202G, Q349R   69 +/− 1.2 326 Q1L, G4C, Q28R, E65K, A72C,E183M, P224L, S311G, D320I, D346E, 69 Q349K, T393V, Y422F, P442S, N445D,R446G, H468L, V482I 327 Q1L, G4C, Q28R, E65V, A72C, D181N, E183M, P224L,F306L, 69 S311G, D320I, D346E, Q349K, T393V, Y422F, P442S, N445D, R446G,H468L, V482I 328 Q1L, G4C, Q28R, E65V, A72C, S86T, D202N, P224L, S311G,N318Y, 69 D320I, D346A, Q349K, T393I, Y422F, N445D, R446G, H468Q, V482T329 Q1L, G4C, Q28K, E65K, A72C, D181N, E183K, S192P, P224L,   69 +/− 0.1S311G, N318Y, D320V, D346E, Q349R, T392M, T393I, Y422F, P442S, H468Q 330Q1L, G4C, Q28K, E65M, A72C, S86T, E183M, H203R, S311D, D320V, 69 T335I,D346E, Q349R, T393A, N445D, R446G, H468Q, V482A 331 Q1L, G4C, Q28R,A72C, D181N, E183V, D202I, H203R, P224L, 69 S311D, D320V, D346V, Q349R,T392M, T393I, N445D, H468L 332 Q1L, G4C, Q28K, W40R, E65K, A72C, S86T,D181N, E183K, S192L, 69 D202I, H203R, S311N, D320N, D346V, Q349K, T392M,T393A, Y422F, N445D, R446S 333 G4C, E65V, A72C, S86T, Y155H, D181N,E183V, Q190K, P224L, 69.1 S311N, D320V, D346V, Q349K, T392M, T393V,Y422F, P442S, N445D, R446G, H468L, V482A 334 Q1L, G4C, Q28R, E65M, A72C,S86T, E183M, D202N, S311D, N318Y, 69.1 D320N, T335I, D346A, Q349R,T392M, T393I, Y422F, P442S, N445D, R446G, H468L, V482A 335 Q1L, G4C,Q28R, E65V, A72C, D181N, E183M, S311N, N318Y, 69.1 D320I, T335I, D346E,Q349K, T393V, Y422F, N445D, R446G, H468L, V482T 336 Q1L, G4C, Q28R,E65K, A72C, S86T, D181N, D202N, S311G, D346E, 69.1 Q349K, T393I, Y422F,N445D, R446G, H468L, V482I 337 Q1L, G4C, Q28R, E65K, A72C, D181N, D202N,S311N, N318Y, 69.1 +/− 0.6 D320I, D346E, Q349K, T393I, Y422F, N445D,R446G, H468L, V482T 338 Q1L, G4C, T7Q, A8S, N10T, S24T, T27Q, Q28R,N29T, D57S, D64N, 69.1 E65V, Q69K, A72C, D181N, P224L, S311D, N318Y,D320N, D346A, Q349K, T392M, T393I, Y422F, N445D, R446G, H468Q, V482I 339Q1L, G4C, Q28R, E65K, A72C, S86T, D202N, S311G, N318Y, D320I, 69.1D346A, Q349K, T393I, Y422F, N445D, R446G, H468L, V482I 340 Q1L, G4C,Q28R, A72C, D181N, S311G, N318Y, D320V, D346E, 69.1 Q349K, T393I, Y422F,P442S, N445D, R446G, H468Q 341 Q1L, G4C, Q28R, E65K, A72C, D181N, E183M,S311G, D320I, 69.1 D346E, Q349K, T393V, Y422F, P442S, N445D, R446G,H468L, V482I 342 Q1L, G4C, Q28R, E65K, A72C, D181N, E183M, S311G, D346E,69.1 Q349K, T393V, Y422F, P442S, N445D, R446G, H468L, V482I 343 Q1L,G4C, Q28R, E65V, A72C, E183M, D202N, S311N, D320I, 69.1 D346E, Q349K,T393V, Y422F, N445D, R446G, H468Q, V482T 344 Q1L, G4C, Q28R, E65K, A72C,E183M, S311G, T335I, D346E, 69.2 Q349K, T393V, Y422F, N445D, R446G,H468Q, V482I 345 Q1L, G4C, Q28R, E65V, A72C, S86T, D181N, D202N, S311N,N318Y, 69.2 T335I, D346E, Q349K, T393V, Y422F, N445D, R446G, H468Q,V482I 346 Q1L, G4C, E65M, A72C, S86T, E183K, D202Y, S311G, D320N, T335I,69.2 D346A, Q349K, T393V, Y422F, R446G, H468Q, V482T 347 Q1L, G4C, Q28R,E65V, A72C, S86T, E183M, D202N, P224L, S331N, 69.2 D320I, T335I, A340S,D346A, Q349K, T393V, Y422F, N445D, R446G, H468Q, V482T 348 Q1L, G4C,Q28K, E65K, A72C, S86T, D181N, E183M, H203R, P224L, 69.2 S311D, D320I,D346E, Q349R, T392M, Y422F, R446G, H468L 349 Q1L, G4C, Q28K, E65V, A72C,S86T, D181N, E183M, P224L, S311G, 69.2 +/− 0.5 D320I, D346E, Q349K,T393V, Y422F, P442S, N445D, R446G, H468Q, V482I 350 Q1L, G4C, Q28K,N29Y, E65K, A72C, D181N, S311G, T335I, D346E, 69.2 +/− 0.1 Q349K, T393V,Y422F, N445D, R446G, H468Q, V482T 351 Q1L, G4C, Q28K, E65M, A72C, E183M,S311G, D346E, Q349K, 69.3 T393V, Y422F, P442S, N445D, R446G, H468L,V482T 352 Q1L, G4C, Q28K, E65V, A72C, S86T, D181N, E183K, D202N, S311N,69.3 N318Y, D320N, T335I, D346A, Q349K, T392M, T393V, P442S, N445D,R446G, H468Q 353 Q1L, G4C, Q28K, E65K, A72C, S86T, D181N, E183M, D202N,S311G, 69.3 N318Y, T335I, D346E, Q349K, T393I, Y422F, N445D, R446G,H468Q, V482I, 354 Q1L, G4C, Q28R, E65K, A72C, E183V, S192T, D202N,S311G, 69.3 +/− 0.3 D320V, D346A, Q349K, T393V, P442S, N445D, H468L,V482A 355 Q1L, G4C, Q28R, E65V, A72C, E183M, D202N, S311N, N318Y, 69.3D320I, T335I, D346E, Q349K, T393V, Y422F, N445D, R446G, H468L, V482T 356Q1L, G4C, S24T, Q28R, E65K, A72C, D202N, S311G, T335I, D346E, 69.3 +/−0.1 Q349K, T393I, Y422F, N445D, R446G, H468Q, V482T 357 Q1L, G4C, A72C,S86T, D181N, S192L, D202N, P224L, S311G, 69.3 N318Y, D320V, T335I,D346E, Q349K, T393I, R446G, H468Q 358 Q1L, G4C, Q28R, E65V, A72C, D181N,P224L, S311D, N318Y, 69.4 +/− 0.5 D320N, D346A, Q349K, T392M, T393I,Y422F, N445D, R446G, H468Q, V482I 359 Q1L, G4C, Q28R, E65M, A72C, D181N,E183M, D202N, P224L, 69.4 S311G, D346A, Q349K, T393I, Y422F, P442S,N445D, R446G, H468L, V482T 360 Q1L, G4C, Q28K, E65V, A72C, S86T, E183V,S192I, D202V, D320I, 69.4 +/− 0.1 T335I, D346V, Q349K, T392M, T393I,Y422F, P442S, N445D, R446G, H468R, V482A 361 Q1L, G4C, Q28K, E65M, A72C,S86T, D181N, E183M, D202N, P224L, 69.4 +/− 0.2 S311G, D320I, T335I,D346E, Q349K, T393V, Y422F, N445D, R446G, H468Q, V482T 362 Q1L, G4C,Q28K, A72C, D181N, D202I, S311D, N318Y, T335I, 69.4 D346E, Q349K, T392M,Y422F, R446G, H468L 363 Q1L, G4C, Q28R, E65V, A72C, D181N, E183M, P224L,S311G, 69.4 D320N, D346E, Q349K, T393V, Y422F, T433S, P442S, N445D,R446G, H468L, V482I 364 Q1L, G4C, Q28K, E65V, A72C, D181N, E183M, D202N,S311G, 69.4 +/− 0.2 N318Y, D346E, Q349K, T393I, Y422F, N445D, R446G,H468Q, V482I 365 Q1L, G4C, Q28K, E65K, A72C, D181N, D202N, S311G, N318Y,69.4 +/− 0.5 D346A, Q349K, T393I, Y422F, N445D, R446G, H468Q, V482T 366Q1L, G4C, Q28R, E65K, A72C, S86T, E183M, D202N, D320I, T335I, 69.4D346A, Q349K, T392M, Y422F, N445D, R446G, H468L, V482T 367 Q1L, G4C,Q28K, E65M, A72C, S86T, E183M, S311N, N318Y, D320I, 69.4 T335I, D346E,Q349K, T393I, Y422F, P442S, H468Q, V482I 368 Q1L, G4C, E65M, A72C, S86T,E183V, S192L, D202I, H203R, P224L, 69.4 +/− 0.1 S311G, N318Y, D320V,D346A, Q349K, P442S, N445D, R446G, H468R, V482A 369 Q1L, G4C, T7Q, A8S,N10T, Q28R, E65V, A72C, D181N, P224L, 69.4 S311D, N318Y, D320N, D346A,Q349K, T392M, T393I, Y422F, N445D, R446G, H468Q, V482I 370 Q1L, G4C,E66K, A72C, S86T, D202V, S311N, D320V, T335I, D346V, 69.4 Q349R, T392M,Y422F, N445D, R446G, H468L 371 Q1L, G4C, Q28R, E65K, A72C, D181N, E183M,P224L, S311G, 69.4 D346E, Q349K, T393V, Y422F, P442S, N445D, R446G,H468L, V482I 372 Q1L, G4C, Q28R, D57S, D64N, E65V, Q69K, A72C, D181N,P224L, 69.4 S311D, N318Y, D320N, D346A, Q349K, T392M, T393I, Y422F,N445D, R446G, H468Q, V482I 373 Q1L, G4C, Q28R, A72C, S86T, E183M, S192T,D202N, H203R, 69.4 P224L, S311N, T335I, D346V, Q349R, T392M, T393V,P442S, N445D, R446G, H468Q, V482A 374 Q1L, G4C, Q28K, E65V, A72C, D181N,D202N, S311N, D346A, 69.4 +/− 0.8 Q349K, T393I, Y422F, N445D, R446G,H468Q, V482I 375 Q1L, G4C, Q28R, E65V, A72C, D181N, E183M, P224L, S311G,D320I, 69.5 D346E, Q349K, T393V, Y422F, P442S, N445D, R446G, H468L,V482I, S485T 376 G4C, Q28K, E65M, A72C, S86T, E183M, D202N, P224L,S311G, 69.5 T335I, D346E, Q349K, T393V, Y422F, P442S, N445D, R446G,H468L, V482I 377 Q1L, G4C, Q28R, E65V, A72C, E183M, P224L, S311G, D320I,D346E, 69.5 +/− 0.4 Q349K, T393V, Y422F, G434(AAATG) P442S, N445D,R446G, T457(AAATT), H468L, V482I 378 G4C, Q28R, E65M, A72C, S86T, D181N,E183M, D202Y, P224L, 69.5 S311N, D346A, Q349K, T393I, Y422F, N445D,R446S, H468Q, V482A 379 Q1L, G4C, Q28K, E65V, A72C, S86T, D181N, E183K,D202N, P224L, 69.5 +/− 1.8 S311G, D320I, D346E, Q349K, T393I, Y422F,P442S, N445D, R446G, H468Q, V482T 380 Q1L, G4C, T27S, Q28R, E65V, A72C,D181N, E183M, P224L, S311G, 69.5 D320I, D346E, Q349K, T393V, Y422F,P442S, N445D, R446G, H468L, V482I 381 Q1L, G4C, Q28R, E65M, A72C, S86T,D181N, E183M, P224L, S311G, 69.5 T335I, D346A, Q349K, T393V, Y422F,P442S, N445D, R446G, H468Q, V482T 382 As1-10ausT.reesei, Q28R, E65V,A72C, D181N, P224L, S311D, 69.5 N318Y, D320N, D346A, Q349K, T392M,T393I, Y422F, N445D, R446G, H468Q, V482I 383 Q1L, G4C, Q28R, E65V, A72C,S86T, D202N, P224L, S311G, D320I, 69.5 D346E, Q349K, T393V, Y422F,N445D, R446G, H468Q, V482I 384 Q1L, G4C, Q28K, E65V, A72C, E183M, D202N,S311G, G320I, 69.6 +/− 0.1 D346E, Q349K, T393V, Y422F, P442S, N445D,R446G, H468L, V482I 385 Q1L, G4C, Q28R, E65K, A72C, D181N, P224L, S311N,N318Y, D320I, 69.6 D346E, Q349K, T393V, Y422F, N445D, R446G, H468L,V482I 386 Q1L, G4C, Q28R, E65M, A72C, S90F, D181N, P224L, S311G, N318Y,69.6 D346E, Q349K, T393I, Y422F, P442S, N445D, R446G, H468Q, V482I 387G4C, Q28R, A72C, S86T, D181N, E183V, S192L, D202V, N246S, 69.6 S311D,N318Y, D320V, D346A, Q349K, T393V, P442S, N445D, H468L, V482A 388 Q1L,G4C, Q28K, E65V, A72C, D181N, E183M, D202N, P224L, 69.6 S311N, N318Y,G320I, T335I, D346A, Q349K, T393I, Y422F, N445D, R446G, H468Q, V482T 389Q1L, G4C, Q28R, A72C, S86T, E183M, S192L, H203R, S311D, 69.6 D320V,T335I, D346V, Q349K, N445D, H468Q, V482A 390 Q1L, G4C, Q28R, T59M, E65K,A68T, A72C, S86T, D181N, S192L, 69.6 D202N, H203R, S311D, D320V, T335I,Q349R, T393A, P442S, N445D, R446G, H468L, V482A 391 Q1L, G4C, Q28R,E65V, A72C, D181N, E183M, D202N, P224L, 69.6 S311G, N318Y, T335I, D346E,Q349K, T393V, Y422F, P442S, N445D, R446G, H468Q, V482I 392 Q1L, G4C,Q28R, E65V, A72C, D181N, E183M, P224L, S311G, D320I, 69.6 D346E, Q349K,K355Q, T393V, Y422F, P442S, N445D, R446G, H468L, V482I 393 Q1L, G4C,Q28R, E65V, A72C, D181N, E183M, P224L, S311G, 69.6 N318Y, T335I, D346A,Q349K, T393V, Y422F, P442S, N445D, R446G, H468L, V482T 394 Q1L, G4C,Q28R, E65V, A72C, D181N, E183M, P224L, S311G, D320I, 69.6 D346E, Q349K,T393V, Y422F, P442S, N445D, R446G, T448A, R453G, H468L, V482I 395 Q1L,G4C, Q28R, E65M, A72C, S86T, D181N, E183M, S311N, N318Y, 69.6 +/− 0.5T335I, D346E, Q349K, T393V, Y422F, N445D, R446G, H468Q, V482I 396 Q1L,G4C, E65M, A72C, E183M, D202I, P224L, S311D, N318Y, 69.6 D320V, T335I,D346A, Q349K, T393A, Y422F, P442S, N445D, R446G, R453G, H468Q, V482I 397Q1L, G4C, Q28R, E65V, A72C, E183M, P224L, S311G, D346E, 69.7 Q349K,T393V, Y422F, P442S, N445D, R446G, H468L, V482I 398 Q1L, G4C, Q28R,E65V, A72C, S86T, D181N, E183M, S311G, N318Y, 69.7 +/− 1 D346A, Q349K,T393V, Y422F, N445D, R446G, H468L, V482T 399 Q1L, G4C, S24T, T27Q, Q28R,N29T, E65V, A72C, D181N, P224L, 69.7 S311D, N318Y, D320N, D346A, Q349K,T392M, T393I, Y422F, N445D, R446G, H468Q, V482I 400 Q1L, G4C, E65V,A72C, D181N, E183M, S192I, P224L, S311G, 69.7 D320V, T335I, D346G,Q349K, T393A, Y422F, P442S, N445D, R446G, H468L, V482T 401 Q1L, G4C,Q28R, E65V, A72C, D181N, E183M, P224L, S311G, D320I, 69.8 +/− 0.8 D346E,Q349K, T393V, Y422F, P442S, N445D, R446G, H468L, V482I 402 Q1L, G4C,Q28R, E65V, A72C, D181N, E183M, D202F, S311G, 69.8 N318Y, T335I, Q349K,T393V, Y422F, P442S, N445D, R446G, H468Q 403 Q1L, G4C, Q28K, E65K, A72C,S86T, D181N, D202N, P224L, S311D, 69.8 N318Y, D320N, D346A, Q349R,T392M, T393I, Y422F, N445D, R446G, H468R 404 Q1L, G4C, Q28R, E65V, A72C,D181N, D202N, S311N, D320I, T335I, 69.8 +/− 0 D346E, Q349K, T393V,Y422F, N445D, R446G, H468Q, V482T 405 Q1L, G4C, Q28K, E65K, A72C, D181N,D202N, P224L, S311G, 69.8 N318Y, T335I, D346E, Q349K, T393V, Y422F,N445D, R446G, H468Q, V482I 406 Q1L, G4C, Q28R, E65V, A72C, D181N, E183M,D202N, P224L, 69.8 S311G, D320I, D346E, Q349K, T393V, Y422F, P442S,N445D, R446G, H468L, V482I 407 Q1L, G4C, Q28R, E65V, A72C, D181N, E183M,D202N, S311N, 69.8 T335I, D346A, Q349K, T393V, Y422F, P442S, N445D,R446G, H468L, V482T 408 Q1L, G4C, Q28K, E65K, A72C, S86T, E183M, P224L,S311G, N318Y, 69.8 D320I, T335I, D346E, Q349K, T393V, Y422F, P442S,N445D, R446G, H468Q, V482T 409 Q1L, G4C, A21T, T26I, Q28R, G30A, E65V,A68T, A72C, Y155C, 69.9 D181N, Q190K, D202N, T229M, S311G, D320I, T335I,D346E, Q349K, T393V, Y422F, N445D, R446G, H468Q, P480S, V482I 410 Q1L,G4C, Q28K, E65M, A72C, D181N, E183M, S311G, T335I, 69.9 D346E, G349K,T393V, Y422F, N445D, R446G, H468L, V482T 411 Q1L, G4C, Q28R, E65V, A72C,D181N, E183M, P224L, S311G, D320I, 69.9 D346E, Q349K, T393V, D404N,Y422F, P442S, N445D, R446G, T451S, H468L, V482I 412 Q1L, G4C, Q28R,E65V, A72C, D181N, E183M, P224L, S311G, D320I, 69.9 D346E, Q349K, T393V,Y422F, P442S, N445D, R446G, P462L, H468L, V482I 413 Q1L, G4C, Q28R,E65V, A72C, E183M, D202N, P224L, S311G, 69.9 N318Y, T335I, D346E, Q349K,T393V, Y422F, N445D, R446G, H468L, V482I 414 Q1L, G4C, Q28R, E65V, A72C,D181N, E183M, P224L, T280A, 69.9 S311G, D320I, D346E, Q349K, T393V,Y422F, P442S, N445D, R446G, H468L, V482I 415 Q1L, G4C, Q28R, E65V, Q69R,A72C, D181N, E183M, P224L, S311G, 69.9 D320I, D346E, Q349K, T393V,Y422F, P442S, N445D, R446G, H468L, V482I 416 Q1L, G4C, Q28R, E65V, A72C,D181N, E183M, P224L, K275E, 69.9 S311G, D320I, D346E, Q349K, T393V,Y422F, P442S, N445D, R446G, H468L, V482I 417 Q1L, G4C, Q28K, E65V, A72C,E183K, D202N, P224L, S311N, D320I, 69.9 D346E, Q349K, T393V, Y422F,P442S, N445D, R446G, H468L, V482T 418 Q1L, G4C, Q28K, E65V, A72C, D181N,E183M, S311G, N318Y, 69.9 +/− 0.2 D320I, D346A, Q349K, T393I, Y422F,N445D, R446G, H468Q, V482T 419 Insertion at position A455(AAAPA), Q1L,G4C, Q28R, E65V, A72C, 70 D181N, E183M, P224L, S311G, D320I, D346E,Q349K, T393V, Y422F, P442S, N445D, R446G, H468L, V482I 420 Q1L, G4C,S24T, T26I, Q28R, N29Y, G30A, E65V, A72C, D181N, 70 E183M, P224L, S311G,D320I, D346E, Q349K, T393V, Y422F, P442S, N445D, R446G, H468L, V482I 422Q1L, G4C, Q28R, E65M, A72C, E183M, S311G, D320I, D346A, 70 Q349K, T393V,Y422F, N445D, R446G, H468Q, V482I 423 Q1L, G4C, Q28R, E65V, A72C, D181N,E183M, D202N, S311G, 70.1 D346E, Q349K, T393I, Y422F, N445D, R446G,H468Q, V482I 424 Q1L, G4C, Q28R, E65V, A72C, E183M, P224L, S311G, D320I,D346E, 70.1 Q349K, T393V, Y422F, P442S, N445D, R446G, H468L, V482I 425Q1L, G4C, Q28K, E65K, A72C, D181N, E183M, S311G, N318Y, 70.1 +/− 0.2D320I, T335I, D346E, Q349K, T393V, Y422F, N445D, R446G, H468Q V482T 426Q1L, G4C, Q28R, E65V, A72C, S311G, N318Y, D320I, T335I, D346E, 70.1Q349K, T393V, Y422F, N445D, R446G, H468L, V482T 427 Q1L, G4C, S24C,Q28R, G30A, E65V, A72C, Y155C, D181N, Q190K, 70.1 D202N, P224L, S311G,T335I, D346E, Q349K, D390E, T393V, Y422F, N445D, R446G, R453G, H468Q,P480S, V482I 428 Q1L, G4C, Q28R, E65V, A72C, D181N, E183M, P224L, S311G,D320I, 70.1 D346E, Q349K, T393V, P402S, Y422F, P442S, N445D, R446G,H468L, V482I 429 Q1L, G4C, Q28R, E65K, A72C, E183M, D202N, P224L, S311N,70.1 N318Y, T335I, D346E, Q349K, T393V, Y422F, P442S, N445D, R446G,H468Q, V482I 430 Q1L, G4C, A21T, T26I, Q28R, N29Y, E65V, A72C, Y155C,D181N, 70.1 D202N, P224L, M234T, S311G, D320I, D346E, Q349K, D390E,T393V, Y422F, N445D, R446G, R453G, H468Q, V482I 431 Q1L, G4C, Q28R,E65K, A72C, S86T, D202N, P224L, S311D, D320N, 70.2 T335I, D346V, Q349K,T392M, T393V, Y422F, P442S, R446S, H468L 432 Q1L, G4C, Q28R, E65V, A72C,S86T, E183K, D202N, S311G, N318Y, 70.2 D320I, D346A, Q349K, T393V,Y422F, P442S, N445D, R446G, H468Q, V482I 433 Q1L, G4C, W40R, E65M, A72C,S86T, E183V, S192L, D202I, H203R, 70.2 +/− 0.3 P224L, S311G, N318Y,D320V, D346A, Q349K, P442S, N445D, R446G, H468R, V482A 434 Q1L, G4C,Q28R, E65V, A72C, D181N, E183M, P224L, S311G, 70.2 D346E, Q349K, T393V,Y422F, P442S, N445D, R446G, H468L, V482I 435 Insertion at positionG434(AAATG) and T457(AAATT), Q1L, G4C, 70.2 +/− 0.2 Q28R, E65V, A72C,D181N, E183M, P224L, S311G, D320I, D346E, Q349K, T393V, Y422F, P442S,N445D, R446G, H468L, V482I 436 Q1L, G4C, Q28R, E65V, A72C, K118Q, D181N,E183M, P224L, 70.3 D247N, S311G, D320I, D346E, Q349K, T393V, Y422F,P442S, N445D, R446G, H468L, V482I 437 Q1L, G4C, Q28R, E65V, A72C, D181N,E183M, D202N, P224L, 70.4 +/− 0 S311N, D320I, D346E, Q349K, T393V,Y422F, N445D, R446G, H468Q, V482I 438 Q1L, G4C, Q28R, E65V, A72C, S86T,D202N, P224L, S311G, N318Y, 70.4 +/− 0.9 T335I, D346E, Q349K, T393V,Y422F, N445D, R446G, H468L, V482T 439 Q1L, G4C, Q28K, E65M, A72C, D181N,E183M, D202N, S311G, 70.4 +/− 0.5 N318Y, D346A, Q349K, T393V, Y422F,P442S, N445D, R446G, H468L, V482T 440 Insertion at position P454(VRPQP),Q1L, G4C, Q28R, E65V, A72C, 70.5 D181N, E183M, P224L, S311G, D320I,D346E, Q349K, T393V, Y422F, P442S, N445D, R446G, H468L, V482I 441 Q1L,G4C, Q28R, A72C, D181N, E183V, S192M, D202N, P224L, 70.5 S311D, N318Y,D320N, D346E, Q349R, T393V, Y422F, P442S, R446G, H468L, P480S 442 Q1L,G4C, Q28R, A72C, S86T, D181N, S192L, D202N, P224L, 70.5 S311G, N318Y,D320V, T335I, D345E, D346A, Q349K, Y422F, P442S, N445D, R446G, H468Q,H505Q 443 Q1L, G4C, Q28R, E65V, A72C, S86T, D181N, E183K, P224L, S311G,70.6 +/− 0 N318Y, D320I, D346A, Q349K, T393V, Y422F, P442S, N445D,R446G, H468L, V482I 444 Q1L, G4C, Q28R, E65M, A72C, D181N, E183K, D202N,P224L, 70.6 +/− 0.6 S311G, D320I, D346E, Q349K, T393V, Y422F, P442S,N445D, R446G, H468Q, V482I 445 Q1L, G4C, Q28R, E65V, A72C, D181N, D202N,S311G, T335I, D346A, 70.7 +/− 0.5 Q349K, T393I, Y422F, P442S, N445D,R446G, H468L, V482I 446 Q1L, G4C, Q28R, E65V, A72C, D181N, E183M, P224L,D247N, 70.7 S311G, D320I, D346E, Q349K, T387S, T393V, Y422F, P442S,N445D, R446G, H468L, V482I 447 Q1L, G4C, Q28R, E65V, A72C, D181N, D202N,P224L, S311G, 70.8 +/− 0.4 D346A, Q349K, T393V, Y422F, N445D, R446G,H468L, V482I 448 insertion at G434(AAATG), Q1L, G4C, Q28R, E65V, A72C,D181N, 70.9 E183M, P224L, S311G, D320I, D346E, Q349K, T393V, Y422F,P442S, N445D, R446G, H468L, V482I 449 insertion at T457(AAATT), Q1L,G4C, Q28R, E65V, A72C, D181N, 70.9 E183M, P224L, S311G, D320I, D346E,Q349K, T393V, Y422F, P442S, N445D, R446G, H468L, V482I 450 Q1L, G4C,Q28K, E65V, A72C, S86T, E183K, S192L, D202N, D320N, 71 T335I, D346A,Q349R, T393A, Y422F, P442S, R446G, H468R, V482A 451 Q1L, G4C, Q28R,E65V, A72C, D181N, E183M, P224L, S236F, 71 S311G, G320I, D346E, Q349K,T393V, Y422F, P442S, N445D, R446G, H468L, V482I 452 Q1L, G4C, Q28R,E65K, A72C, D202I, P224L, S311G, N318Y, D320V, 71.1 T335I, D346A, Q349R,T392M, T393A, N445D, R446G, H468L 453 Q1L, G4C, E65M, A72C, S86T, E183V,D202I, P224L, S311G, N318Y, 71.1 D320V, D346A, Q349K, P442S, N445D,R446G, H468L, V482A 454 Q1L, G4C, Q28R, E65V, A72C, K92R, D181N, E183M,P224L, I277V, 71.1 S311G, D320I, D346E, Q349K, T393V, Y422F, P442S,N445D, R446G, H468L, V482I 455 Q1L, G4C, A72C, S86T, D181N, E183K,D202V, P224L, S311G, 71.1 D320V, D346E, Q349R, T393A, Y422F, P442S,N445D, R446G, H468L 456 Q1L, G4C, Q28K, E65V, A72C, D181N, E183M, D202N,P224L, 71.1 +/− 0 S311G, T335I, D346E, Q349K, T393V, Y422F, P442S,N445D, R446G, H468Q, V482I 457 Q1L, G4C, Q28K, E65V, A72C, S86T, D181N,E183K, S192L, D202V, 71.1 S311G, D320I, D346V, Q349R, T393A, Y422F,P442S, N445D, R446G, H468Q, V482I 458 Q1L, G4C, Q28R, E65V, A72C, S86T,D181N, D202N, P224L, S311N, 71.2 +/− 0 N318Y, D320I, D346A, Q349K,T393V, Y422F, N445D, R446G, H468L, V482T 459 Q1L, G4C, A21T, T26I, Q28R,G30A, E65V, A68T, A72C, D181N, 71.3 E183M, D202N, T229M, S311G, A340S,D346E, Q349K, T393V, Y422F, N445D, R446G, H468Q, V482T

Example 9 Characterization of Variants of the Talaromyces emersoniiCBHI/Trichoderma reesei-CBD Fusion (with 6× His-Tag)

80 mL. of fermentation broth were concentrated to a final volume ofapprox. 1 mL. After determination of protein concentration (Bradfordreagent, Biorad, Germany, Standard is BSA form Sigma-Aldrich, Germany)1.2 mg of protein were purified with the Ni-NTA Spin kit (Qiagen,Germany). The purified CBH1 fraction was subsequently assayed byperforming a hydrolysis reaction on pretreated (acid pretreatment) wheatstraw. 12.5 mg (dry mass) of pre-treated wheat straw is mixed with0.0125 mg of purified CBHI and 40CBU Novo188 (Novozymes, Denmark) per mgof CBH1. 50 mM sodium acetate (Sigma-Aldrich, Germany) is added up to500 μL. The assay is kept at temperatures ranging from 50° C. to 65° C.for 48 hours and analysed by HPLC to determine the temperature dependentglucose content.

Example 10 Hydrolysis of Straw with Seq. ID NO:2 Variant ProteinsExpressed from Trichoderma reesei

Selected cellobiohydrolase sequences (Table 7) are expressed fromTrichoderma reesei as described in Example 5 by replacement of thenative CBHI reading frame with the corresponding sequence fusions to theCBHI signal peptide. Samples of the mutant proteins are isolated fromthe culture supernatant and purified by Ni-NTA affinity chromatography.The characterization of the proteins is done by incubation of theproteins (E) with acid-pretreated straw substrate (S) (2.5% dry mattercontent and E/S=0.1% w/w in the reaction) at temperatures between 50° C.and 75° C. for 48 hours in the presence of a non-limiting amount ofbeta-glucosidase followed by determination of the released glucoseconcentration by HPLC. The results are shown in the Table 7. Asindication for the performance the glucose release after 48 hours at 60°C. was taken and found significantly increased in comparison to the wildtype protein. In the Table 7 the Temperatures where still 50% of themaximum glucose yield is reached are given.

TABLE 7 Performance of Trichoderma expressed Cellobiohydrolases on strawTemperature [° C.] of halve optimum sugar release from acid pretreatedstraw (48 h 0.1% Mutation with respect to Seq. Enzyme to Substrate ratio(40 CBU/mg ID NO: 2 Cellulase BGL added) 1 59.3 ± 0.4 2 , Q1L, G4C,A72C, D181N, E183K, Q349R 61.3 3 , Q1L, G4C, A72C, Y155C, Q349K 64.3 4 ,G4C, Q28K, E65M, A72C, S86T, E183M, 73.0 D202N, P224L, S311G, T335I,D346E, Q349K, T393V, Y422F, P442S, N445D, R446G, H468L, V482I 5 , Q1L,G4C, Q28R, E65V, A72C, D181N, 68.7 P224L, S311D, N318Y, D320N, D346A,Q349K, T392M, T393I, Y422F, N445D, R446G, H468Q, V482I 6 , Q1L, G4C,Q28R, E65V, A72C, D181N, 69.3 E183M, P224L, S311G, D320I, D346E, Q349K,T393V, Y422F, P442S, N445D, R446G, H468L, V482I 7 , Q1L, G4C, Q28R,E65V, A72C, E183M, 69.5 D202N, P224L, S311G, N318Y, T335I, D346E, Q349K,T393V, Y422F, N445D, R446G, H468L, V482I 8 , Q1L, G4C, Q28R, E65V, A72C,S86T, 67.5 E183K, D202N, S311G, N318Y, D320I, D346A, Q349K, T393V,Y422F, P442S, N445D, R446G, H468Q, V482I 9 , Q1L, G4C, Q28R, E65V, A72C,E183M, 68.2 P224L, S311G, D320I, D346E, Q349K, T393V, Y422F, P442S,N445D, R446G, H468L, V482I 10 , Q1L, G4C, Q28R, E65V, A72C, P224L, 68.4S311G, D320I, D346E, Q349K, T393V, Y422F, P442S, N445D, R446G, H468L,V482I 11 , Q1L, G4C, Q28R, E65V, A72C, D202N, 67.8 S311N, D320I, T335I,D346E, Q349K, T393V, Y422F, N445D, R446G, H468Q, V482T 12 , Q1L, G4C,Q28K, E65V, A72C, E183M, 68.0 D202N, S311G, N318Y, D346E, Q349K, T393I,Y422F, N445D, R446G, H468Q, V482I 13 , Q2S, G4C, A6L, T7Q, A8S, N10T,Q28K, 72.0 E65K, A72C, E183M, S311G, N318Y, D320I, T335I, D346E, Q349K,T393V, Y422F, N445D, R446G, H468Q, V482T 14 , Q2S, G4C, A6L, T7Q, A8S,N10T, Q28K, 73.8 E65V, A72C, E183M, D202N, P224L, S311G, T335I, D346E,Q349K, T393V, Y422F, P442S, N445D, R446G, H468Q, V482I 15 , Q2S, G4C,A6L, T7Q, A8S, N10T, Q28R, 73.6 E65V, A72C, P224L, S311D, N318Y, D320N,D346A, Q349K, T392M, T393I, Y422F, N445D, R446G, H468Q, V482I 16 , Q2S,G4C, A6L, T7Q, A8S, N10T, Q28K, 71.9 N29Y, E65K, A72C, S311G, T335I,D346E, Q349K, T393V, Y422F, N445D, R446G, H468Q, V482T 17 , Q1L, G4C,Q28R, E65V, A72C, K159KCGRNK, 70.5 E183M, P224L, S311G, D320I, D346E,Q349K, T393V, Y422F, T433TGAAAT, P442S, N445D, R446G, H468L, V482I 18 ,Q2S, G4C, A6L, T7Q, A8S, N10T, S24T, 72.2 Q28R, E65K, A72C, D202N,S311G, T335I, D346E, Q349K, T393I, Y422F, N445D, R446G, H468Q, V482T 19, Q1L, G4C, Q28R, E65V, A72C, E183M, 69.1 P224L, S311G, D320I, D346E,Q349K, T393V, Y422F, P442S, N445D, R446G, P454PVRPQP, H468L, V482I 20 ,Q1L, G4C, Q28R, E65V, A72C, K159KCGRNK, 68.2 E183M, P224L, S311G, D320I,D346E, Q349K, T393V, Y422F, P442S, N445D, R446G, H468L, V482I 21 , Q1L,G4C, S24T, T26I, Q28R, N29Y, 70.2 G30A, E65V, A68T, A72C, E183M, Q190K,P224L, S311G, D320I, D346E, Q349K, T393V, Y422F, P442S, N445D, R446G,H468L, V482I 22 Seq ID No. 12 58.6 ± 0.4

Example 11 Performance of Seq. ID NO: 5 Variants with ImprovedThermostability

PCR-Techniques were user for the transfer of selected mutations into theSeq. ID NO:5 backbone, as deduced from screened Seq. ID NO: 2 Mutantswith superior performance. Protein expressed from Pichia pastoris wastaken from the culture supernatant and tested for their SubstrateConversion Capacity by the procedure given in example 8. In the tablethe calculated IT50 values for the 4-MUL Substrate are given. Slightlyhigher stability of Proteins without CBD compared to the fusion proteinsare found under these conditions. Results are shown in Table 8.

TABLE 8 Comparison of IT50 Values of SEQ. ID NO: 5 and SEQ. ID NO 2Mutants Mutation Pattern with respect IT50 of the original IT50 of theoriginal to Seq. ID NO: 5 Seq. ID NO: 5 mutant Seq. ID NO: 2 mutant 1 wt61.3 60.4 +/− 0.5 2 , G4C, Q28K, E65M, A72C, S86T, E183M, D202N, 70.069.5 P224L, S311G, T335I, D346E, Q349K, T393V, Y422F 3 , Q1L, G4C, Q28R,E65V, A72C, D181N, E183M, 72.0 69.8 +/− 0.8 P224L, S311G, D320I, D346E,Q349K, T393V, Y422F 4 , Q1L, G4C, Q28K, E65K, A72C, D181N, E183M, 70.370.1 +/− 0.2 S311G, N318Y, D320I, T335I, D346E, Q349K, T393V, Y422F 5 ,Q1L, G4C, Q28K, E65V, A72C, D181N, E183M, 71.9 71.1 +/− 0   D202N,P224L, S311G, T335I, D346E, Q349K, T393V, Y422F 6 Q1L, G4C, Q28R, E65V,A72C, G151GCGRSG, 72.7 68.2 D181N, E183M, P224L, S311G, D320I, D346E,Q349K, T393V, Y422F

Example 12 Influence of Different Cellulose Binding Domains on IT 50Values

To evaluate the interchangeability of the CBDs a stabilized Seq.ID NO: 5mutant (Q1L, G4C, Q28R, E65V, A72C, D181N, E183M, P224L, S311G, D320I,D346E, Q349K, T393V, Y422F) is connected with a series of cellulosebinding domains and linker regions from cellobiohydrolases fromdifferent sources by OE-PCR. The resulting coding sequences (SEQ. IDsNO: 21, 23, 25, 27 and 29) are cloned to a Pichia pastoris expressionvector for the expression of the corresponding fusion proteins accordingto SEQ. IDs NO: 20, 22, 24, 26 and 28). IT50 values for the variants areevaluated as described before and are listed in the Table 9. Only smallinfluences of different CBD modules on the stability are observed.

TABLE 9 Performance of different fusions of CBD with their linkers tostabilized and non-stabilized Seq. ID. NO: 5 variants in comparisonTemperature [° C.] of halve optimum sugar release from cellulose DNAProtein (48 h 0.1% Enzyme to Sequence Sequence Origin of IT50 [° C.]Substrate ratio (40 CBU/ Listing Listing linker and CBD (4-MUL Test) mgCellulase BGL added) Comment 1 1 2 T. reesei 60.4 +/− 0.5 57.4 +/− 0.5non stabilized core (wild type) 2 21 20 C. thermophilum 69.5 +/− 0.565.6 3 23 22 P. chrysosporium 69.3 +/− 0.2 65.6 4 25 24 P. janthinellum68.4 +/− 1.2 65.0 5 27 26 I. lacteus 68.8 +/− 0.1 65.2 6 29 28 T. reesei69.8 +/− 0.8 65.0 +/− 0.7 stabilized core and CBD-linker region

TABLE 10 Sequence Listings (Overview) Nucleic  acid [N]/ Protein SEQID[P] Description ID SEQID NO 01 [N]Coding Sequence for Talaromyces emersonii CBHI/Trichoderma reesei -CBD fusion including the alpha factor signal peptide. SEQID NO 02 [P]Mature Sequence of Talaromyces emersonii CBHI/ CBH-aTrichoderma reesei -CBD SEQID NO 03 [N]Coding sequence of the fusion of CBH-a withTrichoderma reesei CBHI Signal peptide SEQID NO 04 [P]Trichoderma reesei CBHI cellulose binding  domain and linker sequenceSEQID NO 05 [P] Talaromyces emersonii CBHI CBH-b SEQID NO 06 [N]Talaromyces emersonii CBHI SEQID NO 07 [N] Humicola grisea CBHISEQID NO 08 [P] Humicola grisea CBHI CBH-d SEQID NO 09 [N]Thermoascus aurantiacus CBHI SEQID NO 10 [P]Thermoascus aurantiacus CBHI CBH-e SEQID NO 11 [N]Trichoderma reesei CBHI SEQID NO 12 [P] Trichoderma reesei CBHI CBH-cSEQID NO 13 [N] Trichoderma viridae CBHI SEQID NO 14 [P]Trichoderma viridae CBHI CBH-f SEQID NO 15 [N]fusion Humicola grisea CBHI/Trichoderma reesei - CBD SEQID NO 16 [P]fusion Humicola grisea CBHI/Trichoderma reesei - CBH-g CBD SEQID NO 17[N] Coding Sequence for. Talaromyces emersonii CBHI/Trichoderma reesei -CBD including the alpha factor signal peptide and a 6x His Tag. SEQID NO 18 [P]Mature Sequence of Talaromyces emersonii CBHI/ CBH-ahTrichoderma reesei -CBD fusion peptide with 6x  His Tag SEQID NO 19 [N]Humicola grisea CBHI, alternative coding sequence SEQID NO: 20 [N]Oligonucleotide primers used with this invention to 41 SEQID NO 42 [P]Talaromyces emersonii CBHI Mutant withChaetmium thermophilum cellobiohydrolase I CBD with 6x His-TAGSEQID NO 43 [N] Coding Sequence for Talaromyces emersonii CBHIwith 6x His-TAG Mutant with Chaetmiumthermophilum cellobiohydrolase I CBD SEQID NO 44 [P]Talaromyces emersonii CBHI Mutant withPhanerochaete chrysosporium cellobiohydrolase CBD with 6x His-TAGSEQID NO 45 [N] Coding Sequence for Talaromyces emersonii CBHIMutant with Phanerochaete chrysosporiumcellobiohydrolase CBD with 6x His-TAG SEQID NO 46 [P]Talaromyces emersonii CBHI Mutant with Penicilliumjantinellum cellobiohydrolase CBD with 6x His-TAG SEQID NO 47 [N]Coding Sequence for Talaromyces emersonii CBHIMutant with Penicillium jantinellum cellobiohydrolase CBD with 6x His-TAG SEQID NO 48 [P]Talaromyces emersonii CBHI Mutant with Irpexlacteus cellobiohydrolase CBD with 6x His-TAG SEQID NO 49 [N]Coding Sequence for Talaromyces emersonii CBHIMutant with Irpex lacteus cellobiohydrolase CBD with 6x His-TAGSEQID NO 50 [P] Talaromyces emersonii CBHI Mutant with mutatedTrichoderma reesei CBD with 6x His-TAG SEQID NO 51 [N]Coding Sequence for Talaromyces emersonii CBHIMutant with mutated Trichoderma reesei CBD with 6x His-TAG SEQID.NO. 01cagcaggccggcacggcgacggcagagaaccacccgcccctgacatggcaggaatgcaccgcccctgggagctgcaccacccagaacggggcggtcgttcttgatgcgaactggcgttgggtgcacgatgtgaacggatacaccaactgctacacgggcaatacctgggaccccacgtactgccctgacgacgaaacctgcgcccagaactgtgcgctggacggcgcggattacgagggcacctacggcgtgacttcgtcgggcagctccttgaaactcaatttcgtcaccgggtcgaacgtcggatcccgtctctacctgctgcaggacgactcgacctatcagatcttcaagctcctgaaccgcgagttcagctttgacgtcgatgtctccaatcttccgtgcggattgaacggcgctctgtactttgtcgccatggacgccgacggcggcgtgtccaagtacccgaacaacaaggctggtgccaagtacggaaccgggtattgcgactcccaatgcccacgggacctcaagttcatcgacggcgaggccaacgtcgagggctggcagccgtcttcgaacaacgccaacaccggaattggcgaccacggctcctgctgtgcggagatggatgtctgggaagcaaacagcatctccaatgcggtcactccgcacccgtgcgacacgccaggccagacgatgtgctctggagatgactgcggtggcacatactctaacgatcgctacgcgggaacctgcgatcctgacggctgtgacttcaacccttaccgcatgggcaacacttctttctacgggcctggcaagatcatcgataccaccaagcccttcactgtcgtgacgcagttcctcactgatgatggtacggatactggaactctcagcgagatcaagcgcttctacatccagaacagcaacgtcattccgcagcccaactcggacatcagtggcgtgaccggcaactcgatcacgacggagttctgcactgctcagaagcaggcctttggcgacacggacgacttctctcagcacggtggcctggccaagatgggagcggccatgcagcagggtatggtcctggtgatgagtttgtgggacgactacgccgcgcagatgctgtggttggattccgactacccgacggatgcggaccccacgacccctggtattgcccgtggaacgtgtccgacggactcgggcgtcccatcggatgtcgagtcgcagagccccaactcctacgtgacctactcgaacattaagtttggtccgatcggtagcacaggtaatccttcaggtggtaatcctccaggtggaaacagaggaacaacgacaactagaagaccagctactacaactggttcaagtccaggtccaactcaatcacactacggtcaatgtggtggtataggttactctggtcccaccgtttgtgcttctggtactacttgccaagttctgaacccttactactcacagtgtctataatgataa Coding Sequence for CBH-a (mature) SEQID.NO. 02QQAGTATAENHPPLTWQECTAPGSCTTQNGAVVLDANWRWVHDVNGYTNCYTGNTWDPTYCPDDETCAQNCALDGADYEGTYGVTSSGSSLKLNFVTGSNVGSRLYLLQDDSTYQIFKLLNREFSFDVDVSNLPCGLNGALYFVAMDADGGVSKYPNNKAGAKYGTGYCDSQCPRDLKFIDGEANVEGWQPSSNNANTGIGDHGSCCAEMDVWEANSISNAVTPHPCDTPGQTMCSGDDCGGTYSNDRYAGTCDPDGCDFNPYRMGNTSFYGPGKIIDTTKPFTVVTQFLTDDGTDTGTLSEIKRFYIQNSNVIPQPNSDISGVTGNSITTEFCTAQKQAFGDTDDFSQHGGLAKMGAAMQQGMVLVMSLWDDYAAQMLWLDSDYPTDADPTTPGIARGTCPTDSGVPSDVESQSPNSYVTYSNIKFGPIGSTGNPSGGNPPGGNRGTTTTRRPATTTGSSPGPTQSHYGQCGGIGYSGPTVCASGTTCQVLNPYYSQCLMature Sequence of CBH-a SEQID.NO. 03atgtatcggaagttggccgtcatctcggccttcttggccacagctcgtgctcagcaggccggcacggcgacggcagagaaccacccgcccctgacatggcaggaatgcaccgcccctgggagctgcaccacccagaacggggcggtcgttcttgatgcgaactggcgttgggtgcacgatgtgaacggatacaccaactgctacacgggcaatacctgggaccccacgtactgccctgacgacgaaacctgcgcccagaactgtgcgctggacggcgcggattacgagggcacctacggcgtgacttcgtcgggcagctccttgaaactcaatttcgtcaccgggtcgaacgtcggatcccgtctctacctgctgcaggacgactcgacctatcagatcttcaagctcctgaaccgcgagttcagctttgacgtcgatgtctccaatcttccgtgcggattgaacggcgctctgtactttgtcgccatggacgccgacggcggcgtgtccaagtacccgaacaacaaggctggtgccaagcacggaaccgggtattgcgactcccaatgcccacgggacctcaagttcatcgacggcgaggccaacgtcgagggctggcagccgtcttcgaacaacgccaacaccggaattggcgaccacggctcctgccgtgcggagatggatgtctgggaagcaaacagcatctccaatgcggtcactccgcacccgtgcgacacgccaggccagacgatgtgctctggagatgactgcggtggcacatactctaacgatcgctacgcgggaacctgcgatcctgacggctgtgacttcaacccttaccgcatgggcaacacttctttctacgggcctggcaagatcatcgataccaccaagcccttcactgtcgtgacgcagttcctcactgatgatggtacggatactggaactctcagcgagatcaagcgcttctacatccagaacagcaacgtcattccgcagcccaactcggacatcagtggcgtgaccggcaactcgatcacgacggagttctgcactgctcagaagcaggcctttggcgacacggacgacttctctcagcacggtggcctggccaagatgggagcggccatgcagcagggtatggtcctggtgatgagtttgtgggacgactacgccgcgcagatgctgtggttggattccgactacccgacggatgcggaccccacgacccctggtattgcccgtggaacgtgtccgacggacccgggcgtcccatcggatgtcgagtcgcagagccccaactcctacgtgacctactcgaacattaagtttggtccgatcggtagcacaggtaatccttcaggtggtaatcctccaggtggaaacagaggaacaacgacaactagaagaccagctactacaactggttcaagtccaggtccaactcaatcacactacggtcaatgtggtggtataggttactctggtcccactgtctgtgcttctggtactacttgccaagttctgaacccttactactcacagtgtctagcttctgcacatcatcaccaccaccattaaCoding sequence of the fusion of CBH-a with Trichoderma reesei CBHI Signalpeptide SEQID.NO. 04GSTGNPSGGNPPGGNRGTTTTRRPATTTGSSPGPTQSHYGQCGGIGYSGPTVCASGTTCQVLNPYYSQCLTrichoderma reesei CBHI cellulose binding domain and linker sequenceSEQID.NO. 05QQAGTATAENHPPLTWQECTAPGSCTTQNGAVVLDANWRWVHDVNGYTNCYTGNTWDPTYCPDDETCAQNCALDGADYEGTYGVTSSGSSLKLNFVTGSNVGSRLYLLQDDSTYQIFKLLNREFSFDVDVSNLPCGLKGALYFVAMDADGGVSKYPNNKAGAKYGTGYCDSQCPRDLKFIDGEANVEGWQPSSNNANTGIGDHGSCCAEMDVWEANSISNAVTPHPCDTPGQTMCSGDDCGGTYSNDRYAGTCDPDGCDFNPYRMGNTSFYGPGKIIDTTKPFTVVTQFLTDDGTDTGTLSEIKRFYIQNSNVIPQPNSDISGVTGNSITTEFCTAQKQAFGDTDDFSQHGGLAKMGAAMQQGMVLVMSLWDDYAAQMLWLDSDYPTDADPTTPGIARGTCPTDSGVPSDVESQSPNSYVTYSNIKFGPINSTFTASTalaromyces emersonii CBHI sequence (CBH-b) SEQID.NO. 06atgagatttccttcaatttttactgcagttttattcgcagcatcctccgcattagctgctccagtcaacactacaacagaagatgaaacggcacaaattccggctgaagctgtcatcggttacttagatttagaaggggatttcgatgttgctgttttgccattttccaacagcacaaataacgggttattgtttataaatactactattgccagcattgctgctaaagaagaaggggtatctttggataaacgtgaggcggaagcaccctctcagcaggccggcacggcgacggcagagaaccacccgcccctgacatggcaggaatgcaccgcccctgggagctgcaccacccagaacggggcggtcgttcttgatgcgaactggcgttgggtgcacgatgtgaacggatacaccaactgctacacgggcaatacctgggaccccacgtactgccctgacgacgaaacctgcgcccagaactgtgcgctggacggcgcggattacgagggcacctacggcgtgacttcgtcgggcagctccttgaaactcaatttcgtcaccgggtcgaacgtcggatcccgtctctacctgctgcaggacgactcgacctatcagatcttcaagcttctgaaccgcgagttcagctttgacgtcgatgtctccaatcttccgtgcggattgaacggcgctctgtactttgtcgccatggacgccgacggcggcgtgtccaagtacccgaacaacaaggctggtgccaagtacggaaccgggtattgcgactcccaatgcccacgggacctcaagttcatcgacggcgaggccaacgtcgagggctggcagccgtcttcgaacaacgccaacaccggaattggcgaccacggctcctgctgtgcggagatggatgtctgggaagcaaacagcatctccaatgcggtcactccgcacccgtgcgacacgccaggccagacgatgtgctctggagatgactgcggtggcacatactctaacgatcgctacgcgggaacctgcgatcctgacggctgtgacttcaacccttaccgcatgggcaacacttctttctacgggcctggcaagatcatcgataccaccaagcccttcactgtcgtgacgcagttcctcactgatgatggtacggatactggaactctcagcgagatcaagcgcttctacatccagaacagcaacgtcattccgcagcccaactcggacatcagtggcgtgaccggcaactcgatcacgacggagttctgcactgctcagaagcaggcctttggcgacacggacgacttctctcagcacggtggcctggccaagatgggagcggccatgcagcagggtatggtcctggtgatgagtttgtgggacgactacgccgcgcagatgctgtggttggattccgactacccgacggatgcggaccccacgacccctggtattgcccgtggaacgtgtccgacggactcgggcgtcccatcggatgtcgagtcgcagagccccaactcctacgtgacctactcgaacattaagtttggtccgatcaactcgaccttcaccgcttcgtgataaCoding sequence of Talaromyces emersonii CBHI fused to the alpha factorsignal peptide SEQID.NO. 07QQAGTITAENHPRMTWKRCSGPGNCQTVQGEVVIDANWRWLHNNGQNCYEGNKWTSQCSSATDCAQRCALDGANYQSTYGASTSGDSLTLKFVTKHEYGTNIGSRFYLMANQNKYQMFTLMNNEFAFDVDLSKVECGINSALYFVAMEEDGGMASYPSNRAGAKYGTGYCDAQCARDLKFIGGKANIEGWRPSTNDPNAGVGPMGACCAEIDVWESNAYAYAFTPHACGSKNRYHICETNNCGGTYSDDRFAGYCDANGCDYNPYRMGNKDFYGKGKTVDTNRKFTVVSRFERNRLSQFFVQDGRKIEVPPPTWPGLPNSADITPELCDAQFRVFDDRNRFAETGGFDALNEALTIPMVLVMSIWDDHHSNMLWLDSSYPPEKAGLPGGDRGPCPTTSGVPAEVEAQYPDAQVVWSNIRFGPIGSTVNVHumicola grisea CBHI (CBH-d) SEQID.NO. 08atgagatttccttcaatttttactgcagttttattcgcagcatcctccgcattagctgctccagtcaacactacaacagaagatgaaacggcacaaattccggctgaagctgtcatcggttacttagatttagaaggggatttcgatgttgctgttttgccattttccaacagcacaaataacgggttattgtttataaatactactattgccagcattgctgctaaagaagaaggggtatctttggataaacgtgaggcggaagcaccctctcagcaggctggtactattactgctgagaaccacccaagaatgacctggaagagatgctctggtccaggaaactgtcagactgttcagggcgaggttgtgattgacgctaattggagatggttgcacaacaacggccagaactgttacgagggtaacaagtggacctctcagtgttcttctgctaccgactgtgctcagagatgtgctttggacggtgccaactaccagtctacctacggtgcttctacctctggtgactctctgaccctgaagttcgttaccaagcacgagtacggaaccaacatcggctctagattctacctgatggccaaccagaacaagtaccagatgttcaccctgatgaacaacgagttcgcctttgacgttgacctgtctaaggtggagtgcggtatcaactctgccctgtacttcgttgctatggaagaggacggtggaatggcttcttacccatctaacagagccggtgctaagtacggtactggttactgtgacgcccagtgtgctagagacctgaagttcatcggtggaaaggccaacattgagggttggagaccatctaccaacgacccaaacgctggtgttggtccaatgggagcttgttgtgccgagattgatgtgtgggagtctaacgcttacgcctacgcttttaccccacacgcttgcggttctaagaacagataccacatctgcgagaccaacaactgtggtggaacctactctgacgacagattcgctggatactgcgacgctaacggttgtgactacaacccatacagaatgggcaacaaggacttctacggcaagggaaagaccgttgacaccaacagaaagttcaccgtggtgtcgagattcgagagaaacagactgtcgcagttctttgtgcaggacggcagaaagattgaggtcccaccaccaacttggccaggattgccaaactctgccgacattaccccagagttgtgtgacgctcagttcagagtgttcgacgacagaaacagatttgctgagaccggtggttttgacgctttgaacgaggctctgaccattccaatggtgctggtgatgtctatttgggacgaccaccactctaacatgttgtggctggactcttcttacccaccagagaaggctggattgccaggtggtgacagaggaccatgtccaactacttcgggtgttccagctgaggttgaggctcagtacccagacgctcaggttgtgtggtcgaacatcagattcggcccaatcggttctaccgtgaacgtgtaaCoding sequence of Humicola grisea CBHI fused to the alpha factor signalpeptide SEQID.NO. 09heagtvtaenhpsltwqqcssggscttqngkvvidanwrwvhttsgytncytgntwdtsicpddvtcaqncaldgadysgtygvttsgnalrlnfvtqssgknigsrlyllqddttyqifkllgqeftfdvdvsnlpcglngalyfvamdadgnlskypgnkagakygtgycdsqcprdlkfingqnnvegwqpsandpnagvgnhgsscaemdvweansistavtphpcdtpgqtmcqgddcggtysstryagtcdtdgcdfnpyqpgnhsfygpgkivdtsskftvvtqfitddgtpegtlteikrfyvqngkvipqsestisgvtgnsitteyctaqkaafdntgffthgglqkisqalaqgmvlvmslwddhaanmlwldstyptdadpdtpgvargtcpttsgvpadvesqnpnsyviysnikvgpinstftanThermoascus auratiacus CBHZ (CBH-e) SEQID.NO. 10atgagatttccttcaatttttactgcagttttattcgcagcatcctccgcattagctgctccagtcaacactacaacagaagatgaaacggcacaaattccggctgaagctgtcaccggttacttagatttagaaggggatttcgatgttgctgttttgccattttccaacagcacaaataacgggttattgtttataaatactactattgccagcattgctgctaaagaagaaggggtatctttggataaacgtgaggcggaagcaccctctcacgaggccggtaccgtaaccgcagagaatcacccttccctgacctggcagcaatgctccagcggcggtagttgtaccacgcagaatggaaaagtcgttatcgatgcgaactggcgttgggtccataccacctctggatacaccaactgctacacgggcaatacgtgggacaccagtatctgtcccgacgacgtgacctgcgctcagaattgtgccttggatggagcggattacagtggcacctatggtgttacgaccagtggcaacgccctgagactgaactttgtcacccaaagctcagggaagaacattggctcgcgcctgtacctgctgcaggacgacaccacttatcagatcttcaagctgctgggtcaggagtttaccttcgatgtcgacgtctccaatctcccttgcgggctgaacggcgccctctactttgtggccatggacgccgacggcaatttgtccaaataccctggcaacaaggcaggcgctaagtatggcactggttactgcgactctcagtgccctcgggatctcaagttcatcaacggtcaggccaacgttgaaggctggcagccgtctgccaacgacccaaatgccggcgttggtaaccacggttcctcgtgcgctgagatggatgtctgggaagccaacagcatctctactgcggtgacgcctcacccatgcgacacccccggccagaccatgtgccagggagacgactgtggtggaacctactcctccactcgatatgctggtacctgcgacactgatggctgcgacttcaatccttaccagccaggcaaccactcgttctacggccccgggaagatcgtcgacactagctccaaattcaccgtcgtcacccagttcatcaccgacgacgggacaccctccggcaccctgacggagatcaaacgcttctacgtccagaacggcaaggtgatcccccagtcggagtcgacgatcagcggcgtcaccggcaactcaatcaccaccgagtattgcacggcccagaaggcagccttcgacaacaccggcttcttcacgcacggcgggctccagaagatcagtcaggctctggctcagggcatggtcctcgtcatgagcctgtgggacgatcacgccgccaacatgctctggctggacagcacctacccgactgatgcggacccggacacccctggcgtcgcgcgcggcacctgccccacgacctccggcgtcccggccgacgtggagccgcagaaccccaattcatatgttatctactccaacatcaaggtcggacccatcaactcgaccttcaccgccaactaaCoding sequence of Thermoascus auratiacus CBHI fused to the alpha factorsignal peptide SEQID.NO. 11atgagatttccttcaatttttactgcagttttattcgcagcatcctccgcattagctgctccagtcaacactacaacagaagatgaaacggcacaaattccggctgaagctgtcatcggttacttagatttagaaggggatttcgatgttgctgttttgccattttccaacagcacaaataacgggttattgtttataaatactactattgccagcattgctgccaaagaagaaggggtatctttggataaacgtgaggcggaagcaccctcttcagcttgtacactgcaatccgagactcatccacctttaacgtggcaaaagtgtagttctggcggaacttgtactcaacagactggtagtgtcgtgatagatgctaactggagatggacacatgcaacgaactcctcaactaactgctacgatggtaacacctggtcttctacattgtgtcctgacaacgaaacctgcgctaagaactgttgtcttgatggagcagcttacgcaagtacatatggtgtgactacctctggtaacagcctttccattggttttgtaacccagtcggctcagaagaatgttggtgctagattgtacctgatggcttcagacaccacataccaggagtttaccttgttgggaaacgagttctctttcgacgtagatgtgtctcagctaccatgtggattgaatggagccttgtactttgtctcaatggatgcagacggaggtgtttcaaagtaccctactaacacagctggtgctaagtatggaactgg&tactgcgattctcaatgcccaagagacctgaagttcatcaacggacaagctaacgttgaaggttgggaaccttctagcaacaacgcaaacactggaattggtggtcatggttcttgctgttcagagatggacatttgggaagccaactccatcagtgaagctttgactccacatccatgcacaactgtcgggcaagaaatttgcgaaggtgatggttgtggtggcacttactctgataacagatacggcggaacatgtgatccagatggatgtgattggaacccatacagactgggtaacacttcgttttacggaccaggttcttccttcactctagacactacgaagaagttgactgtggtcacccaatttgagacttctggtgccattaaccgatactacgtgcagaacggagttactttccaacagccaaacgctgaattgggtagttactcaggcaacgagcttaacgatgactactgcactgctgaagaagcagaatttggtggatcttccttttcggataagggtggattgacgcagttcaagaaagctacctctggtggaatggttctagtcatgagtctgtgggacgatcactacgctaacatgctttggctggactctacttaccctacaaacgagacatcttctactcctggtgctgtaagaggtagctgttctacatcttctggagttccagcccaagttgagagtcaaagtccaaatgccaaggtcaccttctccaacatcaagttcggaccaattggtagcacaggtaatccttcaggtggtaatcctccaggtggaaacagaggaacaacgacaactagaagaccagctactacaactggttcaagtccaggtccaactcaatcacactacggtcaatgcggtggtataggttactctggtcccactgtttgtgcttctggtactacttgccaagttctgaacccttactactcacagtgtctagcttctgcacaccatcatcatcatcattaatgataaCoding sequence for Trichoderma reesei CBHI (CBH-c), including the alphafactor signal peptide and a 6x His Tag. SEQID.NO. 12QSACTLCSETHPPLTWQKCSSGGTCTQQTGSVVIDANWRWTHATNSSTNCYDGNTWSSTLCPDNETCAKNCCLDGAAYASTYGVTTSGNSLSIGFVTQSAQKNVGARLYLMASDTTYQEFTLLGNEFSFDVDVSQLPCGLNGALYFVSMDADGGVSKYPTNTAGAKYGTGYCDSQCPRDLKFINGQANVEGWEPSSNNANTGIGGHGSCCSEMDIWEANSISEALTPHPCTTVGQEICEGDGCGGTYSDNRYGGTCDPDGCDWNPYRLGNTSFYGPGSSFTLDTTKKLTVVTQFETSGAINRYYVQNGVTFQQPNAELGSYSGNELNDDYCTAEEAEFGGSSFSDKGGLTQFKKATSGGMVLVMSLWDDYYANMLWLDSTYPTNETSSTPGAVRGSCSTSSGVPAQVESQSPNAKVTFSNIKFGPIGSTGNPSGGNPPGGNRGTTTTRRPATTTGSSPGPTQSHYGQCGGIGYSGPTVCASGTTCQVLNPYYSQCLTrichoderma reesei CBHI (CBH-c) SEQID.NO. 13atgagatttccttcaatttttactgcagttttattcgcagcatcctccgcattagctgctccagtcaacactacaacagaagatgaaacggcacaaattccggctgaagctgtcatcggttacttagatttagaaggggatttcgatgttgctgttttgccattttccaacagcacaaataacgggttattgtttataaatactactattgccagcattgctgctaaagaagaaggggtatctttggataaacgtgaggcggaagcaccctctcaatctgcttgcaccttgcagtctgaaactcacccaccattgacctggcagaagtgttcttctggcggtacttgtactcagcagaccggttctgttgttatcgacgccaactggagatggactcacgctaccaactcttctaccaactgctacgacggtaacacttggtcgtctaccttgtgtccagacaacgagacctgtgccaagaactgttgtttggacggtgctgcttacgcttctacctacggtgttaccacctctggtaactcgctgtctatcggtttcgttacccagtctgcccagaaaaatgttggtgccagactgtacttgatggcttctgacaccacctaccaagagtttaccctgctgggtaacgagttctctttcgacgtggacgtttctcaactgccatgtggactgaacggtgccctgtacttcgtttctatggacgctgacggtggtgtttctaagtacccaaccaacaccgctggtgctaaatacggaaccggttactgcgattctcagtgcccaagagacctgaagttcatcaacggacaggctaacgttgaaggatgggagccatcttctaacaacgccaacaccggtattggtggtcacggttcttgctgttctgagatggacatctgggaggccaactctatttctgaggctttgaccccacacccatgtactactgtgggtcaagagatctgtgagggtgatggttgtggtggtacttactcggacaacagatacggtggtacttgtgacccagacggttgtgattgggacccatacagactgggtaacacctctttctacggtccaggatcttcttttaccctggacaccaccaagaagttgaccgttgttacccagtttgagacctctggtgccatcaacagatactacgtgcagaacggtgttactttccagcagccaaacgctgaactgggatcttactctggtaacggactgaacgacgactactgtactgctgaggaagctgagttcggtggttcttctttctctgacaagggtggactgacccagtttaagaaggctacctctggcggaatggtgctggttatgtctttgtgggacgactactacgctaacatgctgtggcttgactctacctacccaactaacgagacctcttctaccccaggtgctgttagaggatcttgctctacctcttctggtgttccagctcaggttgagtctcagtctccaaacgccaaggtgaccttctctaacatcaagttcggtccaatcggttctactggtgacccatctggtggtaacccaccaggtggaaacccacctggtactaccactaccagaagaccagctaccaccactggttcttctccaggtccaacccaatctcactacggtcagtgtggtggtattggttactctggtccaaccgtttgtgcttctggaaccacctgtcaggttctgaacccatactactcgcagtgcctgtaaCoding sequence for Trichoderma viride CBHI, including the alpha factorsignal peptide. SEQID.NO. 14QSACTLQSETHPPLTWQKCSSGGTCTQQTGSVVIDANWRWTHATNSSTNCYDGNTWSSTLCPDNETCAKNCCLDGAAYASTYGVTTSGNSLSIGFVTQSAQKNVGARLYLMASDTTYQEFTLLGNEFSFDVDVSQLPCGLNGALYFVSMDADGGVSKYPTNTAGAKYGTGYCDSQCPRDLKFINGQANVEGWEPSSNNANTGIGGHGSCCSEMDIWEANSISEALTPHPCTTVGQEICEGDGCGGTYSDNRYGGTCDPDGCDWDPYRLGNTSFYGPGSSFTLDTTKKLTVVTQFETSGAINRYYVQNGVTFQQPNAELGSYSGNGLNDDYCTAEEAEFGGSSFSDKGGLTQFKKATSGGMVLVMSLWDDYYANMLWLDSTYPTNETSSTPGAVRGSCSTSSGVPAQVESQSPNAKVTFSNIKFGPIGSTGDPSGGNPPGGNPPGTTTTRRPATTTGSSPGPTQSHYGQCGGIGYSGPTVCASGTTCQVLNPYYSQCLTrichoderma viride CBHI (CBH-f) SEQID.NO. 15atgagatttccttcaatttttactgcagttttattcgcagcatcctccgcattagctgctccagtcaacactacaacagaagatgaaacggcacaaattccggctgaagctgtcatcggttacttagatttagaaggggatttcgatgttgctgttttgccattttccaacagcacaaataacgggttattgtttataaatactactattgccagcattgctgctaaagaagaaggggtatctttggataaacgtgaggcggaagcatgctcgcagcaggctggtacaattactgctgagaaccatccaagaatgacgtggaagagatgtagtggtccaggaaactgtcagactgttcagggtgaggtcgtgatagatgctaactggagatggttgcataacaacggccagaactgctacgagggtaacaagtggacctctcagtgttcttctgctaccgactgcgctcagagatgtgctcttgatggagcaaactaccagagtacatatggtgcttctacctctggtgacagccttaccctgaagtttgtaaccaagcacgagtacggaaccaatatcggttctagattctacctgatggctaaccagaacaagtaccagatgtttaccttgatgaacaacgagttcgccttcgacgtagatctgtctaaggtggagtgtggaatcaattctgccttgtactttgtcgctatggaagaggacggaggtatggcttcttacccttctaacagagctggtgctaagtatggaactggatactgcgatgcccaatgcgctagagacctgaagttcatcggtggaaaggctaacattgaaggttggagaccttctaccaacgacccaaacgctggagttggtccaatgggtgcttgctgtgccgagattgacgigtgggaatctaacgcttacgcctacgcttttactccacatgcttgcggttctaagaacagataccacatttgcgaaaccaacaactgaggtggcacttactctgatgacagattcgctggatactgtgatgctaacggatgtgattacaacccatacagaatgggtaacaaggacttttacggaaagggtaagactgttgacactaacagaaagttcactgtggtctcgagatttgagagaaacagactgtcgcagttctttgtgcaggacggaagaaagattgaggtcccaccaccaacttggccaggattgccaaactctgccgacattaccccagagttgtgcgacgctcagttcagagtgtttgacgacagaaacagatttgctgagaccggtggatttgacgctttgaacgaggctctgaccattccaatggttctagtcatgagtatttgggacgatcaccactctaacatgctttggctggactcttcttaccctccagagaaggctggattgcctggtggtgacagaggtccatgtccaacaacttctggagttccagccgaggttgaggctcaatacccagacgcccaggtcgtgtggtccaacatcagattcggaccaattggaagcttaacaggtaatccttcaggtggtaatcctccaggtggaaacagaggaacaacgacaactagaagaccagctactacaactggttcaagtccaggtccaactcaatcacactacggtcaatgtggtggtataggttactctggtcccactgtttgtgcttctggtactacttgccaagttctgaacccttactactcacagtgtctagcttctgcacaccatcatcatcatcattaaCoding Sequence for Humicola grisea CBHI- Trichoderma reesei CBHI cellulosebinding domain fusion protein including the alpha factor signal peptide anda 6x His Tag. SEQID.NO. 16qqagtitaenhprmtwkrcsgpgncqtvggevvidanwrwlhnngqncyegnkwtsqcssatdcaqrcaldganygstygastsgdsltlkfvtkheygtnigsrfylmanqnkyqmftlmnnefafdvdlskvecginsalyfvameedggmasypsnragakygtgycdaqcardlkfiggkaniegwrpstndpnagvgpmgaccaeidvwesnayayaftphacgsknryhicetnncggtysddrfagycdangcdynpyrmgnkdfygkgktvdtnrkftvvsrfernrlsqffvqdgrkievppptwpglpnsaditpelcdaqfrvfddrnrfaetggfdalnealtipmvlvmsiwddhhsnmlwldssyppekaglpggdrgpcpttsgvpaeveaqypdaqvvwsnirfgpigsltgnpsggnppggnrgttttrrpatttgsspgptqshygqcggigysgptvcasgttcqvlnpyysqclasahhhhhhHumicola grisea CBHI- Trichoderma reesei CBHI cellulose binding domainfusion protein including a 6x His Tag (CBH-g) SEQID.NO. 17atgagatttccttcaatttttactgcagttttatccgcagcatcctccgcattagctgctccagtcaacactacaacagaagatgaaacggcacaaattccggctgaagctgtcatcggttacttagatttagaaggggatttcgatgttgctgttttgccattttccaacagcacaaataacgggttattgtttataaatactactattgccagcattgctgctaaagaagaaggggtatctttggataaacgtgaggcggaagcatgctcgcagcaggccggcacggcgacggcagagaaccacccgcccctgacatggcaggaatgcaccgcccctgggagctgcaccacccagaacggggcggtcgttcttgatgcgaactggcgttgggtgcacgatgtgaacggatacaccaactgctacacgggcaatacctgggaccccacgtactgccctgacgacgaaacctgcgcccagaactgtgcgctggacggcgcggattacgagggcacctacggcgtgacttcgtcgggcagctcctcgaaactcaatttcgtcaccgggtcgaacgtcggatcccgtctctacctgctgcaggacgactcgacctatcagatcttcaagctcctgaaccgcgagttcagctctgacgtcgatgtccccaatcttccgtgcggattgaacggcgctctgtactttgtcgccatggacgccgacggcggcgtgtccaagtacccgaacaacaaggctggtgccaagtacggaaccgggtattgcgactcccaatgcccacgggacctcaagttcatcgacggcgaggccaacgtcgagggctggcagccgtcttcgaacaacgccaacaccggaattggcgaccacggctcctgctgtgcggagatggatgtctgggaagcaaacagcatctccaatgcggtcactccgcacccgtgcgacacgccaggccagacgatgtgctctggagatgactgcggtggcacatactctaacgatcgctacgcgggaacctgcgatcctgacggctgtgacttcaacccttaccgcatgggcaacacttctttctacgggcctggcaagatcatcgataccaccaagcccttcactgtcgtgacgcagttcctcactgatgatggtacggatactggaactctcagcgagatcaagcgcttctacatccagaacagcaacgtcattccgcagcccaactcggacatcagtggcgtgaccggcaactcgatcacgacggagttctgcactgctcagaagcaggcctttggcgacacggacgacttctctcagcacggtggcctggccaagatgggagcggccatgcagcagggtatggtcctggtgatgagtttgtgggacgactacgccgcgcagatgctgtggttggattccgactacccgacggatgcggaccccacgacccctggtattgcccgtggaacgtgtccgacggactcgggcgtcccatcggatgtcgagtcgcagagccccaactcctacgtgacctactcgaacattaagtttggtccgatcggtagcacaggtaatccttcaggtggtaatcctccaggtggaaacagaggaacaacgacaactagaagaccagctactacaactggttcaagtccaggtccaactcaatcacactacggtcaatgtggtggtataggttactctggtcccactgtttgtgcttctggtactacttgccaagttctgaacccttactactcacagtgtctagcctctgcacatcatcaccaccaccactaatgataaCoding sequence for Talaromyces emersonii CBHI/Trichoderma reesei -CBDfusion including the alpha factor signal peptide and a 6x His Tag.SEQID.NO. 18QQAGTATAENHPPLTWQECTAPGSCTTQNGAVVLDANWRWVHDVNGYTNCYTGNTWDPTYCPDDETCAQNCALDGADYEGTYGVTSSGSSLKLNFVTGSNVGSRLYLLQDDSTYQIFKLLNREFSFDVDVSNLPCGLNGALYFVAMDADGGVSKYPNNKAGAKYGTGYCDSQCPRDLKFIDGEANVEGWQPSSNNANTGIGDHGSCCAEMDVWEANSISNAVTPHPCDTPGQTMCSGDDCGGTYSNDRYAGTCDPDGCDFNPYRMGNTSFYGPGKIIDTTKPFTVVTQFLTDDGTDTGTLSEIKRFYIQNSNVIPQPNSDISGVTGNSITTEFCTAQKQAFGDTDDFSQHGGLAKMGAAMQQGMVLVMSLWDDYAAQMLWLDSDYPTDADPTTPGIARGTCPTDSGVPSDVESQSPNSYVTYSNIKFGPIGSTGNPSGGNPPGGNRGTTTTRRPATTTGSSPGPTQSHYGQGGGIGYSGPTVCASGTTCQVLNPYYSQCLASAHHHHHHMature Sequence of Talaromyces emersonii CBHI/Trichoderma reesei -CBDfusion with 6x-His tag (CBH-ah) SEQID.NO 19atggccagcgatctggcacagcaggctggtacaattactgctgagaaccatccaagaatgacgtggaagagatgtagtggtccaggaaactgtcagactgttcagggtgaggtcgtgatagatgctaactggagatggttgcataacaacggccagaactgctacgagggtaacaagtggacctctcagtgttcttctgctaccgactgcgctcagagatgtgctcttgatggagcaaactaccagagtacatatggtgcttctacctctggtgacagccttaccctgaagtttgtaaccaagcacgagtacggaaccaatatcggttctagattctacctgatggctaaccagaacaagtaccagatgtttaccttgatgaacaacgagttcgccttcgacgtagatctgtctaaggtggagtgtggaatcaattctgccctgtactttgtcgctatggaagaggacggaggtatggcttcttacccttctaacagagctggtgctaagtatggaactggatactgcgatgcccaatgcgctagagacctgaagttcatcggtggaaaggctaacattgaaggttggagaccttctaccaacgacccaaacgctggagttggtccaatgggtgcttgctgtgccgagattgacgtgtgggaatctaacgcttacgcctacgcttttactccacatgcttgcggttctaagaacagataccacacttgcgaaaccaacaactgtggtggcacttactctgatgacagattcgctggatactgtgatgctaacggatgtgattacaacccacacagaatgggtaacaaggacttttacggaaagggtaagactgttgacactaacagaaagttcactgtggtctcgagatttgagagaaacagaccgtcgcagttctttgtgcaggacggaagaaagattgaggtcccaccaccaacctggccaggattgccaaactctgccgacattaccccagagttgtgcgacgctcagttcagagtgttcgacgacagaaacagatttgctgagaccggtggatttgacgctttgaacgaggctctgaccattccaatggttctagtcatgagtatttgggacgatcaccactctaacatgctttggctggactcttcttaccctccagagaaggctggattgcctggtggtgacagaggtccatgtccaacaacttctggagttccagccgaggttgaggctcaatacccagacgcccaggtcgtgtggtccaacatcagattcggaccaattggtagcacagtgaatgtggcttctgcacaccatcatcatcatcattgaAlternative coding sequence of Humicola grisea CBHI with signal sequenceSEQID NO 42 [P]LQACTATAENHPPLTWQECTAPGSCTTRNGAVVIMANWRWVHDVNGYTNCYTGNTWDPTYCPDDVTCAQNCCLDGADYEGTYGVTSSGSSLKLNFVTGSNVGSRLYLLQDDSTYQIFKLLNREFSFDVDVSNLPCGLNGALYFVAMDADGGVSKYPNNKAGAKYGTGYCDSQCPRDLKFINGMANVEGWQPSSNNANTGIGDHGSCCAEMDVWEANSISNAVTLHPCDTPGQTMCSGDDCGGTYSNDRYAGTCDPDGCDFNPYRMGNTSFYGPGKIIDTTKPFTVVTQFLTDDGTDTGTLSEIKRFYIQNGNVIPQPNSIISGVTGNSITTEFCTAQKQAFGDTDEFSKHGGLAKMGAAMQQGMVLVMSLIDDYAAQMLWLDSDYPTDADPTVPGIARGTCPTDSGVPSDVESQSPNSYVTFSNIKFGPINSTVPGLDGSTPSNPTATVAPPTSTTTSVRSSTTQISTPTSQPGGCTTQKWGQCGGIGYTGCTNCVAGTTCTELNPWYSQCLASAHHHHHHTalaromyces emersonii CBHI Mutant with Chaetomium thermophilumcellobiohydrolase I CBD with 6x His-TAG SEQID NO 43 [N]ctgcaggcctgcacggcgacggcagagaaccacccgcccctgacatggcaggaatgcaccgcccctgggagctgcaccaccaggaacggggcggtcgttgttgatgcgaactggcgttgggtgcacgatgtgaacggatacaccaactgctacacgggcaatacctgggaccccacgtactgccctgacgacgtaacctgcgcccagaactgttgcctggacggcgcggattacgagggcacctacggcgtgacttcgtcgggcagctccttgaaactcaatttcgtcaccgggtcgaacgtcggatcccgtctctacctgctgcaggacgactcgacctatcagatcttcaagctcctgaaccgcgagttcagctttgacgtcgatgtctccaatcttccgtgcggattgaacggcgctctgtactttgtcgccatggacgccgacggcggcgtgtccaagtacccgaacaacaaggctggtgccaagtacggaaccgggtattgcgactcccaatgcccacgggacctcaagttcatcaacggcatggccaacgtcgagggctggcagccgtcatcgaacaacgccaacaccggaattggcgaccacggctcctgctgtgcggagatggatgtctgggaagcaaacagcatctccaatgcggtcactctgcacccgtgcgacacgccaggccagacgatgtgctctggagatgactgcggtggcacatactctaacgatcgctacgcgggaacctgcgatcctgacggctgtgacttcaacccttaccgcatgggcaacacttctttctacgggcctggcaagatcatcgataccaccaagcccttcactgtcgtgacgcagttcctcactgatgatggtacggatactggaactctcagcgagatcaagcgcttctacatccagaacggcaacgtcattccgcagcccaactcgatcatcagtggcgtgaccggcaactcgatcacgacggagttctgcactgctcagaagcaggcctttggcgacacggacgaattctctaagcacggtggcctggccaagatgggagcggccatgcagcagggtatggtcctggtgatgagtttgtgggacgactacgccgcgcagatgctgtggttggattccgactacccgacggatgcggaccccacggtccctggtattgcccgtggaacgtgtccgacggactcgggcgtcccatcggatgtcgagtcgcagagccccaactcctacgtgaccttctcgaacattaagtttggtccgatcaactcgaccgtccctggcctcgacggcagcacccccagcaacccgaccgccaccgttgctcctcccacttctaccaccaccagcgtgagaagcagcactactcagatttccaccccgactagccagcccggcggctgcaccacccagaagtggggccagtgcggtggtatcggctacaccggctgcactaactgcgttgctggcactacctgcactgagctcaacccctggtacagccagtgcctggcttctgctcatcatcaccatcaccacCoding Sequence for Talaromyces emersonii CBHI Mutant with Chaetmiumthermophilum cellobiohydrolase I CBD with 6x His-TAG SEQID NO 44 [P]LQACTATAENHPPLTWQECTAPGSCTTRNGAVVLDANWRWVHDVNGYTNCYTGNTWDPTYCPDDVTCAQNCCLDGADYEGTYGVTSSGSSLKLNFVTGSNVGSRLYLLQDDSTYQIFKLLNREFSFDVDVSNLPCGLNGALYFVAMDADGGVSKYPNNKAGAKYGTGYCDSQCPRDLKFINGMANVEGWQPSSNNANTGIGDHGSCCAEMDVWEANSISNAVTLHPCDTPGQTMCSGDDCGGTYSNDRYAGTCDPDGCDFNPYRMGNTSFYGPGKIIDTTKPFTVVTQFLTDDGTDTGTLSEIKRFYIQNGNVIPQPNSIISGVTGNSITTEFCTAQKQAFGDTDEFSKHGGLAKMGAAMQQGMVLVMSLWDDYAAQMLWLDSDYPTDADPTVPGIARGTCPTDSGVPSDVESQSPNSYVTFSNIKFGPINSTYTGTVSSSSVSSSHSSTSTSSSHSSSSTPPTQPTGVTVPQWGQCGGIGYTGSTTCASPYTCHVLNPYYSQCYASAHHHHHHTalaromyces emersonii CBHI Mutant with Phanerochaete chrysosporiumcellobiohydrolase CBD with 6x His-TAG SEQID NO 45 [N]ctgcaggcctgcacggcgacggcagagaaccacccgcccctgacatggcaggaatgcaccgcccctgggagctgcaccaccaggaacggggcggtcgttcttgatgcgaactggcgttgggtgcacgatgtgaacggatacaccaactgctacacgggcaatacctgggaccccacgtactgccctgacgacgtaacctgcgcccagaactgttgcctggacggcgcggattacgagggcacctacggcgtgacttcgtcgggcagctccttgaaactcaatttcgtcaccgggtcgaacgtcggatcccgtctctacctgctgcaggacgactcgacctatcagatcttcaagctcctgaaccgcgagttcagctttgacgtcgatgtctccaatcttccgtgcggattgaacggcgctctgcactttgtcgccatggacgccgacggcggcgtgtccaagtacccgaacaacaaggctggtgccaagtacggaaccgggtattgcgactcccaatgcccacgggacctcaagttcatcaacggcatggccaacgtcgagggctggcagccgccatcgaacaacgccaacaccggaattggcgaccacggctcctgctgtgcggagatggatgtctgggaagcaaacagcatctccaatgcggtcactctgcacccgtgcgacacgccaggccagacgatgtgctctggagatgactgcggtggcacatactctaacgatcgctacgcgggaacctgcgatcctgacggctgtgacttcaacccttaccgcatgggcaacacttctttctacgggcctggcaagatcatcgataccaccaagcccttcactgtcgtgacgcagttcctcactgatgatggtacggatactggaactctcagcgagatcaagcgcttctacatccagaacggcaacgtcattccgcagcccaactcgatcatcagtggcgtgaccggcaactcgatcacgacggagttctgcactgctcagaagcaggcctttggcgacacggacgaattctctaagcacggtggcctggccaagatgggagcggccatgcagcagggtatggtcctggtgatgagtttgtgggacgactacgccgcgcagatgctgtggttggattccgactacccgacggatgcggaccccacggtccctggtatcgcccgtggaacgtgtccgacggactcgggcgtcccatcggatgtcgagtcgcagagccccaactcctacgtgaccttctcgaacattaagtttggtccgatcaactcgacctacactggaactgtttcttcatcctccgtttcatcttctcactcctccacttctacttcatcttcccattcctcatcttccactccaccaactcaaccaactggtgttactgttccacaatggggacaatgtggtggtattggttacactggttccactacttgtgcttccccatactcttgtcacgttttgaacccatactactcccaatgttacgcttctgctcatcatcaccatcaccactaaCoding Sequence for Talaromyces emersonii CBHI Mutant with Phanerochaetechrysosporium cellobiohydrolase CBD with 6x His-TAG SEQID NO 46 [P]lqactataenhppltwqectapgscttrngavvldanwrwvhdvngytcnytgntwdptycpddvtcaqnccldgadyegtygvtssgeslklnfvtgsnvgsrlyllqddstyqifkllnrefsfdvdvsnlpcglngalyfvamdadggvskypnnkagakygtgycdsgcprdlkfingmanvegwqpssnnantgigdhgsccaemdvweanaisnavtlhpcdtpgqtmcsgddcggtysndryagtcdpdgcdfnpyrmgntsfygpgkiidttkpftvvtqfltddgtdtgtlseikrfyiqngnvipqpnsiisgvtgnsittefctaqkqafgdtdefskhgglakmgaamqqgmvlvmslwddyaaqmlwldsdyptdadptvpgiargtcptdsgvpsdvesqspnsyvtfsnikfgpinstftggttsssstttttskststsssskttttsvtttttssgssgtgaahwaqcggngwtgpttcvspytctkqndwysqclasahhhhhhTalaromyces emersonii CBHI Mutant with Penicillium janthinellumcellobiohydrolase CBD with 6x His-TAG SEQID NO 47 [N]ctgcaggcctgcacggcgacggcagagaaccacccgcccctgacatggcaggaatgcaccgcccctgggagctgcaccaccaggaacggggcggtcgttcttgatgcgaactggcgttgggtgcacgatgtgaacggatacaccaactgctacacgggcaatacctgggaccccacgtactgccctgacgacgtaacctgcgcccagaactgttgcctggacggcgcggattacgagggcacctacggcgtgacttcgtcgggcagctccttgaaactcaatttcgtcaccgggtcgaacgtcggatcccgtctctacctgctgcaggacgactcgacctatcagatcttcaagctcctgaaccgcgagttcagctttgacgtcgatgtctccaatcttccgtgcggattgaacggcgctctgtactttgtcgccatggacgccgacggcggcgtgtccaagtacccgaacaacaaggctggtgccaagtacggaaccgggtattgcgactcccaatgcccacgggacctcaagttcatcaacggcatggccaacgtcgagggctggcagccgtcatcgaacaacgccaacaccggaattggcgaccacggctcctgctgtgcggagatggatgtctgggaagcaaacagcatctccaatgcggtcactctgcacccgtgcgacacgccaggccagacgatgtgctctggagatgactgcggtggcacatactctaacgatcgctacgcgggaacctgcgatcctgacggctgtgacttcaacccttaccgcatgggcaacacttctttctacgggcctggcaagatcatcgataccaccaagcccttcactgtcgtgacgcagttcctcactgatgatggtacggatactggaactctcagcgagatcaagcgcttctacatccagaacggcaacgtcattccgcagcccaactcgatcatcagtggcgtgaccggcaactcgatcacgacggagttctgcactgctcagaagcaggcctctggcgacacggacgaattctctaagcacggtggcctggccaagatgggagcggccatgcagcagggtatggtcctggtgatgagtttgtgggacgactacgccgcgcagatgctgtggttggattccgactacccgacggatgcggaccccacggtccctggtattgcccgtggaacgtgtccgacggactcgggcgtcccatcggatgtcgagtcgcagagccccaactcctacgtgaccttctcgaacattaagtttggtccgatcaactcgaccttcactggtggtactacttcatcctcctccactaccactacaacttccaagtccacttccacttcatcttcatccaagactacaactacttccgttacaactactactacttcctctggttcttctggtactggtgctgctcattgggctcaatgtggtggtaatggatggactggtccaactacttgtgtttccccatacacttgtactaagcagaacgactggtactctcaatgtttggcttctgctcatcatcaccatcaccacCoding Sequence for Talaromyces emersonii CBHI Mutant with Penicilliumjanthinellum cellobiohydrolase CBD with 6x His-TAG SEQID NO 48 [P]lqactataenhppltwqectapgscttrngavvldanwrwvhdvngytncytgntwdptycpddvtcaqnccldgadyegtygvtssgsslklnfvtgsnvgsrlyllqddstyqifkllnrefsfdvdvsnlpcglngalyfvamdadggvskypnnkagakygtgycdsqcprdlkfingmanvegwqpssnnantgigdhgsccaemdvweansisnavtlhpcdtpgqtmcsgddcggtysndryagtcdpdgcdfnpyrmgntsfygpgkiidttkpftvvtqfltddgtdtgtlseikrfyiqngnvipqpnsiisgvtgnsittefctaqkqafgdtdefskhgglakmgaamqqgmvlvmslwddyaaqmlwldsdyptdadptvpgiargtcptdsgvpsdvesqspnsyvtfsnikfgpinstftgtgstspsspagpvssatsvasqptqpaqgtvaqwgqcggtgftgptvcaspftchvvnpyysqcyasahhhhhhTalaromyces emersonii CBHI Mutant with Irpex lacteus cellobiohydrolase CBDwith 6x His-TAG SEQID NO 49 [N]ctgcaggcctgcacggcgacggcagagaaccacccgcccctgacatggcaggaatgcaccgcccctgggagctgcaccaccaggaacggggcggtcgttcttgatgcgaactggcgttgggcgcacgatgtgaacggatacaccaactgctacacgggcaatacctggg&ccccacgtactgccctgacgacgtaacctgcgcccagaactgttgcctggacggcgcggattacgagggcacctacggcgtgacttcgtcgggcagccccttgaaactcaatttcgtcaccgggtcgaacgtcggatcccgtctctacctgctgcaggacgactcgacctatcagatcttcaagctcccgaaccgcgagttcagctttgacgtcgatgtctccaatcttccgtgcggattgaacggcgctctgtactttgtcgccatggacgccgacggcggcgtgtccaagtacccgaacaacaaggctggtgccaagtacggaaccgggtattgcgacccccaatgcccacgggacctcaagttcatcaacggcatggccaacgtcgagggctggcagccgtcatcgaacaacgccaacaccggaattggcgaccacggctcccgctgtgcggagatggatgtctgggaagcaaacagcatctccaatgcggtcactctgcacccgtgcgacacgccaggccagacgatgtgctctggagatgactgcggtggcacatactctaacgatcgctacgcgggaacctgcgatcctgacggctgtgacttcaacccttaccgc&tgggcaacacttctttctacgggcctggcaagatcatcgataccaccaagcccttcactgtcgtgacgcagttcctcactgatgatggtacggatactggaactctcagcgagatcaagcgcttctacatccagaacggcaacgtcattccgcagcccaactcgatcatcagtggcgtgaccggcaactcgatcacgacggagttctgcactgctcagaagcaggcctttggcgacacggacgaattctctaagcacggtggcctggccaagatgggagcggccatgcagcagggtatggtcctggtgatgagtttgtgggacgactacgccgcgcagatgctgtggttggattccgactacccgacggatgcggaccccacggtccctggtattgcccgtggaacgtgtccgacggactcgggcgtcccatcggatgtcgagtcgcagagccccaactcctacgtgaccttctcgaacattaagtttggtccgatcaactcgaccttcactggtactggttctacttctccatcttctccagctggtccagtttcttcttccacttccgttgcttcccaaccaactcaaccagctcaaggtactgttgctcaatggggacaatgtggtggtactggtttcactggtccaactgtttgtgcttccccattcacttgtcacgttgttaacccatactacccccagtgttacgcttctgctcatcatcatcaccatcacCoding Sequence for Talaromyces emersonii CBHI Mucant with Irpex lacteuscellobiohydrolase CBD witb 6x His-TAG SEQID NO 50 [P]lgactataenhppltwqectapgscttrngavvldanwrwvhdvngytncytgntwdptycpddvtcaqnccldgadyegtygvtssgsslklnfvtgsnvgsrlyllqddstyqifkllnrefsfdvdvsnlpcglngalyfvamdadggvskypnnkagakygcgycdsqcprdlkfingmanvegwqpssnnantgigdhgsccaemdvweansisnavtlhpcdtpgqtmcsgddcggtysndryagtcdpdgcdfnpyrmgntsfygpgkiidttkpftvvtqfltddgtdtgtlseikrfyiqngnvipqpnsiisgvtgnsittefctaqkqafgdtdetskhgglakmgaamqqgmvlvmslwddyaaqmlwldsdyptdadptvpgiargtcptdsgvpsdvesqspnsyvtfsnikfgpigstgnpsggnpsggdggttttrrpatttgsspgptqslygqcggigysgpticasgttcqvlnpyysqclasahhhhhhTalaromyces emersonii CBHI Mutant with mutated Trichoderma reesei CBD with6x His-TAG SEQID NO 51 [N]ctgcaggcctgcacggcgacggcagagaaccacccgcccctgacatggcaggaatgcaccgcccctgggagctgcaccaccaggaacggggcggtcgttcttgatgcgaactggcgttgggtgcacgatgtgaacggatacaccaactgctacacgggcaatacctgggaccccacgtactgccctgacgacgtaacctgcgcccagaactgttgcctggacggcgcggattacgagggcacctacggcgtgacttcgtcgggcagctccttgaaactcaatttcgtcaccgggtcgaacgtcggatcccgtatctacctgctgcaggacgactcgacctatcagatcttcaagctcctgaaccgcgagttcagctttgacgtcgatgtctccaatcttccgtgcggattgaacggcgctctgtactttgtcgccatggacgccgacggcggcgtgtccaagtacccgaacaacaaggctggtgccaagtacggaaccgggtattgcgactcccaatgcccacgggacctcaagttcatcaacggcatggccaacgtcgagggctggcagccgtcatcgaacaacgccaacaccggaattggcgaccacggctcctgctgtgcggagatggatgtctgggaagcaaacagcatctccaatgcggtcactctgcacccgtgcgacacgccaggccagacgatgtgctctggagatgactgcggtggcacatactctaacgatcgctacgcgggaacctgcgatcctgacggctgtgacttcaacccttaccgcatgggcaacacttctttctacgggcctggcaagatcatcgataccaccaagcccttcactgtcgtgacgcagttcctcactgatgatggtacggatactggaactctcagcgagatcaagcgcttctacatccagaacggcaacgtcattccgcagcccaactcgatcatcagtggcgtgaccggcaactcgatcacgacggagttctgcactgctcagaagcaggcctttggcgacacggacgaattctctaagcacggtggcctggccaagatgggagcggccatgcagcagggtatggtcctggtgatgagtttgtgggacgactacgccgcgcagatgctgtggttggattccgactacccgacggatgcggaccccacggtccctggtattgcccgtggaacgtgtccgacggactcgggcgtcccatcggatgtcgagtcgcagagccccaactcctacgtgaccttctcgaacattaagtttggtccgatcggtagcacaggtaatccttcaggtggtaatccttcaggtggagacggcggaacaacgacaactagaagaccagctactacaactggttcaagtccaggtccaactcaatcactatacggtcaatgtggtggtataggttactctggtcccactatttgtgcttctggtactacttgccaagttctgaacccttactactcacagtgtctagcttctgcacatcatcaccaccaccatCoding Sequence for Talaromyces emersonii CBHI Mutant with mutatedTrichoderma reesei CBD with 6x His-TAG

What is claimed:
 1. A recombinant polypeptide having cellobiohydrolaseactivity, wherein the recombinant polypeptide comprises at least 90%sequence identity to SEQ ID NO: 5, wherein the recombinant polypeptideis modified at one or more positions selected from the group consistingof Q1, Q2, T7, A8, N10, Q28, E65, S86, D181, E183, D202, P224, S311,N318, T335, D346, Q349, T392, T393, and Y422, and wherein thepolypeptide maintains an IT50 for 60 minutes at a temperature of 62° C.or higher.
 2. The recombinant polypeptide according to claim 1,comprising additional substitution(s) deletion(s) or insertion(s) whilemaintaining at least 90% sequence identity to SEQ ID NO:
 5. 3. Therecombinant polypeptide according to claim 1, comprising additionalmodification at one or more of the following amino acid residues bysubstitution or deletion at positions G4, P12, T15, A21, G23, S24, T26,T27, N29, G30, A31, V32, N37, W40, V41, G46, Y47, T48, N49, C50, T52,N54, D57, T59, Y60, D64, A68, Q69, A72, V84, S89, S90, K92, S99, Q109,D110, D111, I116, F117, K118, L119, L120, D120, V130, G139, A145, M146,V152, K154, Y155, N157, N158, K159, K163, G167, Q172, F179, I180, E187,G188, Q190, S192, S193, N194, I200, H203, D211, V212, A221, D228, T229,G231, T233, M234, S236, T243, Y244, S245, N246, D247, G251, F260, G266,K275, I276, I277, T280, L290, D293, G294, T295, T297, T299, S301, K304,F306, N310, V313, I314, D320, I321, T325, N327, A340, F341, D343, T344,D345, H350, A354, K355, A358, Q361, Q362, G363, M364, V367, D373, Y374,A375, A376, P386, T387, D390, T392, P394, T400, P402, T403, D404, D410,N417, S418, T421, F427, P429, I430, G431, T433, G434, N435, P436, S437;or additional one or more insertion group(s) after positions G151, orK159, each said insertion group(s) comprising five amino acids, whilemaintaining at least 90% sequence identity to SEQ ID NO:
 5. 4. Therecombinant polypeptide according to claim 1, comprising additionalmodification at one or more amino acid residues as follows: PositionModified to: G4 C A6 G, L, or V P12 Q T15 S A21 S, T, or C G23 A, D, orN S24 T, C, or N T26 I, or N T27 S, or Q N29 T, or Y G30 A A31 S V32 GN37 S W40 R V41 T G46 S Y47 S, or F T48 A N49 S C50 S T52 D N54 S D57 ST59 M Y60 H D64 N A68 T Q69 K, or R A72 V, or C V84 A S89 N S90 T, or FK92 R S99 T Q109 R D110 G, S, or N D111 H, or E I116 V, K, or E F117 YK118 A, T, or Q L119 L, or I L120 P, or M D129 N V130 I G139 S A145 TM146 C G151 GCGRSG V152 A, or E K154 R Y155 S, C, H N157 S N158 D K159E, KCGRNK K163 C G167 C Q172 Q F179 I I180 N E187 K G188 C Q190 L, or KS192 L, I, P, T, or M S193 L, P, or T N194 G, L, I, V, S, C, K, R, D, Q,or Y I200 N or F H203 R D211 G V212 L A221 V D228 N T229 A, S, or M G231D T233 S M234 L, I, V, T, or K S236 F, or Y T243 G, A, L, I, V, P, S, C,M, R, D, Q, F, Y, or W Y244 H, F S245 T N246 S, K, or D D247 N G251 RF260 C G266 S K275 E I276 V I277 V T280 A L290 H D293 R, or H G294 AT295 S T297 N T299 I, or S S301 C K304 R F306 L, Y N310 D, E V313 I I314F D320 I, V, E, N I321 N T325 A, or I N327 Y A340 G, S, or T F341 C D343A T344 M D345 E H350 Y A354 T K355 Q A358 E Q361 R Q362 G, R, or H G363P M364 L, or S V367 A D373 E Y374 A, P, S, C, R, H, D A375 G, L, V, T,C, M, R, D, E, N, Q, or Y A376 T P386 L, S T387 A, S D390 G, E P394 CT400 S P402 S T403 K D404 N D410 G N417 Y S418 P T421 I F427 Y P429 CI430 L G431 D T433 S, or E G434 S, or GAAATG N435 Q P436 S S437 P

while maintaining at least 90% sequence identity to SEQ ID NO:
 5. 5. Therecombinant polypeptide according to claim 1, which is expressed andsecreted at a level of more than 100 mg/1 into a supernatant of a yeastculture medium after introduction of a nucleic acid encoding therecombinant polypeptide according to claim 1 into the yeast.
 6. Therecombinant polypeptide according to claim 1, wherein the one or moremodifications are indicated in the following table: Position Modifiedto: Q1 L Q2 P, or S T7 Q A8 S N10 T, or D Q28 L, K , R, or N E65 V, M,or K S86 T D181 N E183 V, M, or K D202 G, I, V, N, F, or Y P224 L S311G, D, or N N318 I, H, D, or Y T335 I D346 G, A, V, or E Q349 K, or RT392 S, M, or K T393 A, I, V, or S Y422 F.


7. A method of producing a recombinant polypeptide, comprising thesteps: a. obtaining a host cell: b. cultivating the host cell underconditions which the recombinant polypeptide according to claim 1 isexpressed; and c. recovering the recombinant polypeptide according toclaim
 1. 8. A process of enzymatically degrading lignocellulosic biomasscomprising, degrading lignocellulosic biomass by the recombinantpolypeptide according to claim 1, or processing textiles by therecombinant polypeptide according to claim 1 or adding the recombinantpolypeptide according to claim 1 as ingredient in detergents or addingthe recombinant polypeptide according to claim 1 as ingredient in foodor feed compositions.